Biallelic markers for use in constructing a high density disequilibrium map of the human genome

ABSTRACT

The present invention relates to genomic maps comprising biallelic markers, new biallelic markers, and methods of using biallelic markers. Primers hybridizing to regions flanking these biallelic markers are also provided. This invention provides polynucleotides and methods suitable for genotyping a nucleic acid containing sample for one or more biallelic markers of the invention. Further, the invention provides a number of methods utilizing the biallelic markers of the invention including methods to detect a statistical correlation between a biallelic marker allele and a phenotype and/or between a biallelic marker haplotype and a phenotype.

RELATED APPLICATIONS

The present application is a Continuation-in-Part application of U.S. patent application Ser. No. 09/298,850, filed Apr. 21, 1999, now abandoned, and International Patent Application No. PCT/IB99/00822, filed Apr. 21, 1999, which both claim priority to U.S. Provisional Patent Application Serial No. 60/082,614, filed Apr. 21, 1998 and U.S. Provisional Patent Application Serial No. 60/109,732, filed Nov. 23, 1998, the disclosures of each of which are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

Recent advances in genetic engineering and bioinformatics have enabled the manipulation and characterization of large portions of the human genome. While efforts to obtain the full sequence of the human genome are rapidly progressing, there are many practical uses for genetic information which can be implemented with partial knowledge of the sequence of the human genome.

As the full sequence of the human genome is assembled, the partial sequence information available can be used to identify genes responsible for detectable human traits, such as genes associated with human diseases, and to develop diagnostic tests capable of identifying individuals who express a detectable trait as the result of a specific genotype or individuals whose genotype places them at risk of developing a detectable trait at a subsequent time. Each of these applications for partial genomic sequence information is based upon the assembly of genetic and physical maps which order the known genomic sequences along the human chromosomes.

The present invention relates to an ordered set of human genomic sequences comprising single nucleotide polymorphisms, as well as the use of these polymorphisms as a high resolution map of the human genome, methods of identifying genes associated with detectable human traits, and diagnostics for identifying individuals who carry a gene which causes them to express a detectable trait or which places them at risk of expressing a detectable trait in the future.

Advantages of the Biallelic Markers of the Present Invention

The map-related biallelic markers of the present invention offer a number of important advantages over other genetic markers such as RFLP (Restriction fragment length polymorphism), VNTR (Variable Number of Tandem Repeats) markers and earlier STS-(sequence tagged sites) derived markers.

The first generation of markers, RFLPs, are variations that modify the length of a restriction fragment. However, methods used to identify and type RFLPs are relatively wasteful of materials, effort, and time. Since they are biallelic markers (they present only two alleles, the restriction site being either present or absent), their maximum heterozygosity is 0.5. The theoretical number of RFLPs distributed along the entire human genome is more than 10⁵, which leads to a potential average inter-marker distance of 30 kilobases. However, in reality, the number of evenly distributed RFLPs which occurs at a sufficient frequency in the population to make them useful for tracking of genetic polymorphisms is very limited.

The second generation of genetic markers were VNTRs, which can be categorized as either minisatellites or microsatellites. Minisatellites are tandemly repeated DNA sequences present in units of 5-50 repeats which are distributed along regions of the human chromosomes ranging from 0.1 to 20 kilobases in length. Since they present many possible alleles, their informative content is very high. Minisatellites are scored by performing Southern blots to identify the number of tandem repeats present in a nucleic acid sample from the individual being tested. However, there are only 10⁴ potential VNTRs that can be typed by Southern blotting. Thus, the number of easily typed informative markers in these maps is far too small for the average distance between informative markers to fulfill the requirements for a useful genetic map. Moreover, both RFLP and VNTR markers are costly and time-consuming to develop and assay in large numbers.

Initial attempts to construct genetic maps based on non-RFLP biallelic markers have focused on identifying biallelic markers lying within sequence tagged sites (STS), pieces of genomic DNA having a known sequence and averaging about 250 bases in length. More than 30,000 STSs have been identified and ordered along the genome (Hudson et al., Science 270:1945-1954 (1995); Schuler et al., Science 274:540-546 (1996), the disclosures of which are incorporated herein by reference in their entireties). For example, the Whitehead Institute and Genethon's integrated map contains 15,086 STSs.

These sequence tagged sites can be screened to identify polymorphisms, preferably Single Nucleotide Polymorphisms (SNPs), more preferably non RFLP biallelic markers therein. Generally polymorphisms are identified by determining the sequence of the STSs in 5 to 10 individuals.

Wang et al. (Cold Spring Harbor Laboratory: Abstracts of Papers Presented on Genome Mapping and Sequencing p. 17 (May 14-18, 1997), the disclosure of which is incorporated herein by reference in its entirety) recently announced the identification and mapping of 750 Single Nucleotide Polymorphisms issued from the sequencing of 12,000 STSs from the Whitehead/MIT map, in eight unrelated individuals. The map was assembled using a high throughput system based on the utilization of DNA chip technology available from Affymetrix (Chee et al., Science 274:610-614 (1996), the disclosure of which is incorporated herein by reference in its entirety).

However, according to experimental data and statistical calculations, less than one out of 10 of all STSs mapped today will contain an informative Single Nucleotide Polymorphism. This is primarily due to the short length of existing STSs (usually less than 250 bp). If one assumes 10⁶ informative SNPs spread along the human genome, there would on average be one marker of interest every 3×10⁹/10⁶, i.e. every 3,000 bp. The probability that one such marker is present on a 250 bp stretch is thus less than {fraction (1/10)}.

While it could produce a high density map, the STS approach based on currently existing markers does not put any systematic effort into making sure that the markers obtained are optimally distributed throughout the entire genome. Instead, polymorphisms are limited to those locations for which STSs are available.

The even distribution of markers along the chromosomes is critical to the future success of genetic analyses. In particular, a high density map having appropriately spaced markers is essential for conducting association studies on sporadic cases, aiming at identifying genes responsible for detectable traits such as those which are described below.

As will be further explained below, genetic studies have mostly relied in the past on a statistical approach called linkage analysis, which took advantage of microsatellite markers to study their inheritance pattern within families from which a sufficient number of individuals presented the studied trait. Because of intrinsic limitations of linkage analysis, which will be further detailed below, and because these studies necessitate the recruitment of adequate family pedigrees, they are not well suited to the genetic analysis of all traits, particularly those for which only sporadic cases are available (e.g. drug response traits), or those which have a low penetrance within the studied population.

Association studies enabled by the biallelic markers of the present invention offer an alternative to linkage analysis. Combined with the use of a high density map of appropriately spaced, sufficiently informative markers, association studies, including linkage disequilibrium-based genome wide association studies, will enable the identification of most genes involved in complex traits.

Single nucleotide polymorphism or biallelic markers can be used in the same manner as RFLPs and VNTRs but offer several advantages. Single nucleotide polymorphisms are densely spaced in the human genome and represent the most frequent type of variation. An estimated number of more than 10⁷ sites are scattered along the 3×10⁹ base pairs of the human genome. Therefore, single nucleotide polymorphisms occur at a greater frequency and with greater uniformity than RFLP or VNTR markers which means that there is a greater probability that such a marker will be found in close proximity to a genetic locus of interest. Single nucleotide polymorphisms are less variable than VNTR markers but are mutationally more stable.

Also, the different forms of a characterized single nucleotide polymorphism, such as the biallelic markers of the present invention, are often easier to distinguish and can therefore be typed easily on a routine basis. Biallelic markers have single nucleotide based alleles and they have only two common alleles, which allows highly parallel detection and automated scoring. The biallelic markers of the present invention offer the possibility of rapid, high-throughput genotyping of a large number of individuals.

Biallelic markers are densely spaced in the genome, sufficiently informative and can be assayed in large numbers. The combined effects of these advantages make biallelic markers extremely valuable in genetic studies. Biallelic markers can be used in linkage studies in families, in allele sharing methods, in linkage disequilibrium studies in populations, in association studies of case-control populations. An important aspect of the present invention is that biallelic markers allow association studies to be performed to identify genes involved in complex traits. Association studies examine the frequency of marker alleles in unrelated case- and control-populations and are generally employed in the detection of polygenic or sporadic traits. Association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families (linkage studies). Biallelic markers in different genes can be screened in parallel for direct association with disease or response to a treatment. This multiple gene approach is a powerful tool for a variety of human genetic studies as it provides the necessary statistical power to examine the synergistic effect of multiple genetic factors on a particular phenotype, drug response, sporadic trait, or disease state with a complex genetic etiology.

The present invention relates to a high density linkage disequilibrium-based genetic maps of the human genome which comprise the map-related biallelic markers of the invention and will allow the identification of genes responsible for detectable traits using genome-wide association studies and linkage disequilibrium mapping.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of a set of novel map-related biallelic markers (See Table 1). The position of these markers and knowledge of the surrounding sequence have been used to design polynucleotide compositions which are useful in high density mapping of the human genome as well as in determining the identity of nucleotides at the marker position, and more complex association and haplotyping studies which are useful in determining the genetic basis for disease states. In addition, the compositions and methods of the invention find use in the identification of the targets for the development of pharmaceutical agents and diagnostic methods, as well as in the characterization of the differential efficacious responses to and side effects from pharmaceutical agents acting on a disease as well as other treatments.

A first embodiment of the present invention is a map of the human genome comprising an ordered array of biallelic markers, wherein at least 1, 2, 5, 10, 20, 25, 30, 50, 100, 200, 500, 1000, 2000 or 3000 of said biallelic markers are map-related biallelic markers. In addition, the maps of the present invention encompass maps with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said map-related biallelic marker may be selected individually or in any combination from the group consisting of the biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908 and the complements thereof; optionally said ordered array comprises at least 20,000, 40,000, 60,000, 80,000, 100,000, or 120,000 biallelic markers; optionally, wherein said biallelic markers are separated from one another by an average distance of 10 kb-200 kb, 15 kb-150 kb, 20 kb-100 kb, 100 kb-150 kb, 50-100 kb, or 25 kb-50 kb in the human genome; optionally, said biallelic markers are distributed at an average density of at least one biallelic marker every 150 kb, 50 kb, or 30 kb in the human genome; or optionally, wherein, all of said biallelic markers are selected to have a heterozygosity rates of at least about 0.18, 0.32, or 0.42.

A second embodiment of the invention encompasses isolated, purified or recombinant polynucleotides consisting of, consisting essentially of, or comprising a contiguous span of nucleotides of a sequence selected as an individual or in any combination from the group consisting of SEQ ID No. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908, 3935 to 7842, 7866 to 11773, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 10125, 10126 to 11599, and 11600 to 11773, or the complements thereof, wherein said contiguous span is at least 8, 10, 12, 15, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides in length, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID. The present invention also relates to polynucleotides hybridizing under stringent or intermediate conditions to a sequence selected from the group consisting of SEQ ID No. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908, 3935 to 7842, 7866 to 11773, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 10125, 10126 to 11599, and 11600 to 11773 and the complements thereof. In addition, the polynucleotides of the invention encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: said contiguous span may optionally comprise a map-related biallelic marker; optionally either the 1st or the 2nd allele of the respective SEQ ID No., as indicated in Table 1, may be specified as being present at said map-related biallelic marker; optionally, said biallelic marker may be within 6, 5, 4, 3, 2, or 1 nucleotides of the center of said polynucleotide or at the center of said polynucleotide; optionally, said polynucleotide may comprise, consist of, or consist essentially of a contiguous span which ranges in length from 8, 10, 12, 15, 18 or 20 to 21, 25, 35, 40, 43, or 47 nucleotides; optionally, said polynucleotide may comprise, consist of, or consist essentially of a contiguous span which ranges in length from 8, 10, 12, 15, 18 or 20 to 21, 25, 35, 40, 43, or 47 nucleotides, or be specified as being 12, 15, 18, 20, 25, 35, 40, 43, or 47 nucleotides in length and including an map-related biallelic marker of said sequence, and optionally the 1st allele of Table 1 is present at said biallelic marker; optionally, the 3′ end of said contiguous span may be present at the 3′ end of said polynucleotide; optionally, biallelic marker may be present at the 3′ end of said polynucleotide; optionally, the 3′ end of said polynucleotide may be located within or at least 2, 4, 6, 8, or 10 nucleotides upstream of a map-related biallelic marker in said sequence, to the extent that such a distance is consistent with the lengths of the particular Sequence ID; optionally, the 3′ end of said polynucleotide may be located 1 nucleotide upstream of a map-related biallelic marker in said sequence; and optionally, said polynucleotide may further comprise a label.

A third embodiment of the invention encompasses any polynucleotide of the invention attached to a solid support. In addition, the polynucleotides of the invention which are attached to a solid support encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said polynucleotides may be specified as attached individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, 25, 50, 100, 200, or 500 distinct polynucleotides of the inventions to a single solid support; optionally, polynucleotides other than those of the invention may attached to the same solid support as polynucleotides of the invention; optionally, when multiple polynucleotides are attached to a solid support they may be attached at random locations, or in an ordered array; optionally, said ordered array may be addressable.

A fourth embodiment of the invention encompasses the use of any polynucleotide for, or any polynucleotide for use in, determining the identity of nucleotides at a map-related biallelic marker. In addition, the polynucleotides of the invention for use in determining the identity of nucleotides at a map-related biallelic marker encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said map-related biallelic marker may be selected individually or in any combination from the group consisting of the biallelic markers of SEQ ID No. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908 and the complements thereof; optionally, said polynucleotide may comprise a sequence disclosed in the present specification; optionally, said polynucleotide may comprise, consist of, or consist essentially of any polynucleotide described in the present specification; optionally, said determining may be performed in a hybridization assay, sequencing assay, microsequencing assay, or an enzyme-based mismatch detection assay; optionally, said polynucleotide may be attached to a solid support, array, or addressable array; optionally, said polynucleotide may be labeled.

A fifth embodiment of the invention encompasses the use of any polynucleotide for, or any polynucleotide for use in, amplifying a segment of nucleotides comprising a map-related biallelic marker. In addition, the polynucleotides of the invention for use in amplifying a segment of nucleotides comprising a map-related biallelic marker encompass polynucleotides with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said map-related biallelic marker may be selected individually or in any combination from the group consisting of the biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908 and the complements thereof; optionally, said polynucleotide may comprise, consist of, consist essentially of, or comprise a sequence selected individually or in any combination from the group consisting of SEQ ID Nos. 3935 to 7842, 7866 to 11773, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 10125, 10126 to 11599, and 11600 to 11773; optionally, said polynucleotide may comprise, consist of, or consist essentially of any polynucleotide described in the present specification; optionally, said amplifying may be performed by a PCR or LCR. Optionally, said polynucleotide may be attached to a solid support, array, or addressable array. Optionally, said polynucleotide may be labeled.

A sixth embodiment of the invention encompasses methods of genotyping a biological sample comprising determining the identity of a nucleotide at a map-related biallelic marker. In addition, the genotyping methods of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said map-related biallelic marker may be selected individually or in any combination from the group consisting of the biallelic markers of SEQ ID No. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908 and the complements thereof; optionally, said method further comprises determining the identity of a second nucleotide at said biallelic marker, wherein said first nucleotide and second nucleotide are not base paired (by Watson & Crick base pairing) to one another; optionally, said biological sample is derived from a single individual or subject; optionally, said method is performed in vitro; optionally, said biallelic marker is determined for both copies of said biallelic marker present in said individual's genome; optionally, said biological sample is derived from multiple subjects or individuals; optionally, said method further comprises amplifying a portion of said sequence comprising the biallelic marker prior to said determining step; optionally, wherein said amplifying is performed by PCR, LCR, or replication of a recombinant vector comprising an origin of replication and said portion in a host cell; optionally, wherein said determining is performed by a hybridization assay, sequencing assay, microsequencing assay, or an enzyme-based mismatch detection assay.

A seventh embodiment of the invention comprises methods of estimating the frequency of an allele in a population comprising genotyping individuals from said population for a map-related biallelic marker and determining the proportional representation of said biallelic marker in said population. In addition, the methods of estimating the frequency of an allele in a population of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said map-related biallelic marker may be selected individually or in any combination from the group consisting of the biallelic markers of SEQ Nos. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908 and the complements thereof; optionally, determining the frequency of a biallelic marker allele in a population may be accomplished by determining the identity of the nucleotides for both copies of said biallelic marker present in the genome of each individual in said population and calculating the proportional representation of said nucleotide at said map-related biallelic marker for the population; optionally, determining the frequency of a biallelic marker allele in a population may be accomplished by performing a genotyping method on a pooled biological sample derived from a representative number of individuals, or each individual, in said population, and calculating the proportional amount of said nucleotide compared with the total.

An eighth embodiment of the invention comprises methods of detecting an association between an allele and a phenotype, comprising the steps of a) determining the frequency of at least one map-related biallelic marker allele in a trait positive population, b) determining the frequency of said map-related biallelic marker allele in a control population and; c) determining whether a statistically significant association exists between said genotype and said phenotype. In addition, the methods of detecting an association between an allele and a phenotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said map-related biallelic marker may be selected individually or in any combination from the group consisting of the biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908 and the complements thereof, optionally, said control population may be a trait-negative population, or a random population; optionally, wherein said phenotype is selected from the group consisting of disease, treatment response, treatment efficacy, drug response, drug efficacy, and drug toxicity; optionally, the determining steps a) and b) are performed on all of the biallelic markers of SEQ ID Nos. 1 to 3908.

An ninth embodiment of the present invention encompasses methods of estimating the frequency of a haplotype for a set of biallelic markers in a population, comprising the steps of: a) genotyping each individual in said population for at least one map-related biallelic marker, b) genotyping each individual in said population for a second biallelic marker by determining the identity of the nucleotides at said second biallelic marker for both copies of said second biallelic marker present in the genome; and c) applying a haplotype determination method to the identities of the nucleotides determined in steps a) and b) to obtain an estimate of said frequency. In addition, the methods of estimating the frequency of a haplotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally said haplotype determination method is selected from the group consisting of asymmetric PCR amplification, double PCR amplification of specific alleles, the Clark method, or an expectation maximization algorithm; optionally, said map-related biallelic marker may be selected individually or in any combination from the group consisting of the biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908 and the complements thereof; optionally, said second biallelic marker is a map-related biallelic marker; optionally, the identity of the nucleotides at the biallelic markers in every one of the sequences of SEQ ID No. 1 to 3908 is determined in steps a) and b).

A tenth embodiment of the present invention encompasses methods of detecting an association between a haplotype and a phenotype, comprising the steps of: a) estimating the frequency of at least one haplotype in a trait positive population according to a method of estimating the frequency of a haplotype of the invention; b) estimating the frequency of said haplotype in a control population according to the method of estimating the frequency of a haplotype of the invention; and c) determining whether a statistically significant association exists between said haplotype and said phenotype. In addition, the methods of detecting an association between a haplotype and a phenotype of the invention encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said map-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID No. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908 and the complements thereof; optionally, said control population may be a trait-negative population, or a random population; optionally, wherein said phenotype is selected from the group consisting of disease, treatment response, treatment efficacy, drug response, drug efficacy, and drug toxicity; optionally, the identity of the nucleotides at the biallelic markers in every one of the following sequences: SEQ ID No. 1 to 3908 is included in the estimating steps a) and b).

An eleventh embodiment of the present invention is a method of identifying a gene associated with a detectable trait comprising the steps of: a) determining the frequency of each allele of at least one map-related biallelic marker in individuals having the detectable trait and individuals lacking the detectable trait; b) identifying at least one alleles of one or biallelic markers having a statistically significant association with the detectable trait; and c) identifying a gene in linkage disequilibrium with said allele. In addition, the methods of the present invention for identifying a gene associated with a detectable trait encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, wherein the method further comprises d) identifying a mutation in the gene identified in step c) which is associated with the detectable trait; optionally, wherein the individuals having the detectable trait and the individuals lacking the detectable trait are readily distinguishable from one another; optionally, wherein the individuals having the detectable trait and the individuals lacking the detectable trait are selected from a bimodal population; optionally, wherein the individuals having the detectable trait are at one extreme of the population and the individuals lacking the detectable trait are at the other extreme of the population; optionally, said map-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID No. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908 and the complements thereof; optionally, wherein said detectable trait is selected from the group consisting of disease, treatment response, treatment efficacy, drug response, drug efficacy, and drug toxicity.

A twelfth embodiment of the present invention is a method of identifying biallelic markers associated with a detectable trait comprising the steps of: a) determining the frequencies of a set of biallelic markers comprising at least one map-related biallelic marker in individuals who express said detectable trait and individuals who do not express said detectable trait; and b) identifying one or more biallelic markers in said set which are statistically associated with the expression of said detectable trait. In addition, the methods of the present invention for identifying biallelic markers associated with a detectable trait encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said map-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID No. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908 and the complements thereof; optionally, wherein said detectable trait is selected from the group consisting of disease, treatment response, treatment efficacy, drug response, drug efficacy, and drug toxicity.

A thirteenth embodiment of the present invention is a method of identifying biallelic marker(s) in linkage disequilibrium with a trait causing allele or in linkage disequilibrium with a trait-associated biallelic marker comprising the steps of: a) selecting at least one map-related biallelic marker which is in the genomic region suspected of containing the trait-causing allele or the trait-associated biallelic marker; and b) determining which of the map-related biallelic markers are associated with the trait-causing allele or in linkage disequilibrium with the trait-associated biallelic marker. In addition, the methods of the present invention for identifying biallelic marker(s) in linkage disequilibrium with a trait causing allele or in linkage disequilibrium with a trait-associated biallelic marker encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said map-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID No. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908 and the complements thereof; optionally, wherein said detectable trait is selected from the group consisting of disease, treatment response, treatment efficacy, drug response, drug efficacy, and drug toxicity.

A fourteenth embodiment of the present invention is a method for determining whether an individual is at risk of developing a detectable trait or suffers from a detectable trait comprising the steps of: a) obtaining a nucleic acid sample from the individual; b) screening the nucleic acid sample with at least one map-related biallelic marker; and c) determining whether the nucleic acid sample contains at least one allele of said map-related biallelic marker statistically associated with the detectable trait. In addition, the methods of the present invention for determining whether an individual is at risk of developing a detectable trait or suffers from a detectable trait encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said map-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID No. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908 and the complements thereof; optionally, wherein said detectable trait is selected from the group consisting of disease, treatment response, treatment efficacy, drug response, drug efficacy, and drug toxicity.

A fifteenth embodiment of the present invention is a method of administering a drug or a treatment comprising the steps of: a) obtaining a nucleic acid sample from an individual; b) determining the identity of the polymorphic base of at least one map-related biallelic marker which is associated with a positive response to the treatment or the drug; or at least one biallelic map-related marker which is associated with a negative response to the treatment or the drug; and c) administering the treatment or the drug to the individual if the nucleic acid sample contains said biallelic marker associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug. In addition, the methods of the present invention for administering a drug or a treatment encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said map-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID No. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908 and the complements thereof; or optionally, the administering step comprises administering the drug or the treatment to the individual if the nucleic acid sample contains said biallelic marker associated with a positive response to the treatment or the drug and the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug.

A sixteenth embodiment of the present invention is a method of selecting an individual for inclusion in a clinical trial of a treatment or drug comprising the steps of: a) obtaining a nucleic acid sample from an individual; b) determining the identity of the polymorphic base of at least one map-related biallelic marker which is associated with a positive response to the treatment or the drug, or at least one map-related biallelic marker which is associated with a negative response to the treatment or the drug in the nucleic acid sample, and c) including the individual in the clinical trial if the nucleic acid sample contains said map-related biallelic marker associated with a positive response to the treatment or the drug or if the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug. In addition, the methods of the present invention for selecting an individual for inclusion in a clinical trial of a treatment or drug encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said map-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID No. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908 and the complements thereof; optionally, the including step comprises administering the drug or the treatment to the individual if the nucleic acid sample contains said biallelic marker associated with a positive response to the treatment or the drug and the nucleic acid sample lacks said biallelic marker associated with a negative response to the treatment or the drug.

A seventeenth embodiment of the present invention is a method of identifying a gene associated with a detectable trait comprising the steps of: a) selecting a gene suspected of being associated with a detectable trait; and b) identifying at least one map-related biallelic marker within said gene which is associated with said detectable trait. In addition, the methods of the present invention for identifying a gene associated with a detectable trait encompass methods with any further limitation described in this disclosure, or those following, specified alone or in any combination: optionally, said map-related biallelic marker may be in a sequence selected individually or in any combination from the group consisting of SEQ ID No. 1 to 3908, 1 to 2260, 2261 to 3734, 3734 to 3908 and the complements thereof; optionally, the identifying step comprises determining the frequencies of the map-related biallelic marker(s) in individuals who express said detectable trait and individuals who do not express said detectable trait and identifying one or more biallelic markers which are statistically associated with the expression of the detectable trait.

Additional embodiments are set forth in the Detailed Description of the Invention and in the Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a cytogenetic map of chromosome 21.

FIG. 2a shows the results of a computer simulation of the distribution of inter-marker spacing on a randomly distributed set of biallelic markers indicating the percentage of biallelic markers which will be spaced a given distance apart for 1, 2, or 3 markers/BAC in a genomic map (assuming a set of 20,000 minimally overlapping BACs covering the genome are evaluated).

FIG. 2b shows the results of a computer simulation of the distribution of inter-marker spacing on a randomly distributed set of biallelic markers indicating the percentage of biallelic markers which will be spaced a given distance apart for 1, 3, or 6 markers/BAC in a genomic map (assuming a set of 20,000 minimally overlapping BACs covering the genome are evaluated).

FIG. 3 shows, for a series of hypothetical sample sizes, the p-value significance obtained in association studies performed using individual markers from the high-density biallelic map, according to various hypotheses regarding the difference of allelic frequencies between the trait-positive and trait-negative samples.

FIG. 4 is a hypothetical association analysis conducted with a map comprising about 3,000 biallelic markers.

FIG. 5 is a hypothetical association analysis conducted with a map comprising about 20,000 biallelic markers.

FIG. 6 is a hypothetical association analysis conducted with a map comprising about 60,000 biallelic markers.

FIG. 7 is a haplotype analysis using biallelic markers in the Apo E region.

FIG. 8 is a simulated haplotype analysis using the biallelic markers in the Apo E region included in the haplotype analysis of FIG. 7.

FIG. 9 shows a minimal array of overlapping clones which was chosen for further studies of biallelic markers associated with prostate cancer, the positions of STS markers known to map in the candidate genomic region along the contig, and the locations of biallelic markers along the BAC contig harboring a genomic region harboring a candidate gene associated with prostate cancer which were identified using the methods of the present invention.

FIG. 10 is a rough localization of a candidate gene for prostate cancer which was obtained by determining the frequencies of the biallelic markers of FIG. 9 in affected and unaffected populations.

FIG. 11 is a further refinement of the localization of the candidate gene for prostate cancer using additional biallelic markers which were not included in the rough localization illustrated in FIG. 10.

FIG. 12 is a haplotype analysis using the biallelic markers in the genomic region of the gene associated with prostate cancer.

FIG. 13 is a simulated haplotype using the six markers included in haplotype 5 of FIG. 12.

FIG. 14 is a block diagram of an exemplary computer system.

FIG. 15 is a flow diagram illustrating one embodiment of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database.

FIG. 16 is a flow diagram illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous.

FIG. 17 is a flow diagram illustrating one embodiment of an identifier process 300 for detecting the presence of a feature in a sequence.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

SEQ ID Nos. 1 to 3908 contain nucleotide sequences comprising a portion of the map-related biallelic markers of the invention.

SEQ ID Nos. 3909 to 3934 contain nucleotide sequences comprising a portion of the map-related biallelic markers which are shown to be associated with Alzheimer's disease, prostate cancer or asthma as described in the Examples.

SEQ ID Nos. 3935 to 7842 contain nucleotide sequences of upstream amplification primers (PU) designed to amplify sequences containing the biallelic markers of SEQ ID Nos. 1 to 3908.

SEQ ID Nos. 7843 to 7865 contain nucleotide sequences of upstream amplification primers (PU) designed to amplify sequences containing the biallelic markers of SEQ ID Nos. 3909 to 3934.

SEQ ID Nos. 7866 to 11773 contain nucleotide sequences of downstream amplification primers (RP) designed to amplify sequences containing the biallelic markers of SEQ ID Nos. 1 to 3908.

SEQ ID Nos. 11774 to 11796 contain nucleotide sequences of downstream amplification primers (RP) designed to amplify sequences containing the biallelic markers of SEQ ID Nos. 3909 to 3934.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Before describing the invention in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used to describe the invention herein.

Definitions

As used interchangeably herein, the terms “nucleic acids” “oligonucleotides”, and “polynucleotides” include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. The term “nucleotide” as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form. The term “nucleotide” is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide. Although the term “nucleotide” is also used herein to encompass “modified nucleotides” which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, for examples of analogous linking groups, purine, pyrimidines, and sugars see for example PCT publication No. WO 95/04064. However, the polynucleotides of the invention are preferably comprised of greater than 50% conventional deoxyribose nucleotides, and most preferably greater than 90% conventional deoxyribose nucleotides. The polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.

The term “purified” is used herein to describe a polynucleotide or polynucleotide vector of the invention which has been separated from other compounds including, but not limited to other nucleic acids, carbohydrates, lipids and proteins (such as the enzymes used in the synthesis of the polynucleotide), or the separation of covalently closed polynucleotides from linear polynucleotides. A polynucleotide is substantially pure when at least about 50%, preferably 60 to 75% of a sample exhibits a single polynucleotide sequence and conformation (linear versus covalently close). A substantially pure polynucleotide typically comprises about 50%, preferably 60 to 90% weight/weight of a nucleic acid sample, more usually about 95%, and preferably is over about 99% pure. Polynucleotide purity or homogeneity may be indicated by a number of means well known in the art, such as agarose or polyacrylamide gel electrophoresis of a sample, followed by visualizing a single polynucleotide band upon staining the gel. For certain purposes higher resolution can be provided by using HPLC or other means well known in the art.

The term “primer” denotes a specific oligonucleotide sequence which is complementary to a target nucleotide sequence and used to hybridize to the target nucleotide sequence. A primer serves as an initiation point for nucleotide polymerization catalyzed by either DNA polymerase, RNA polymerase or reverse transcriptase.

The term “probe” denotes a defined nucleic acid segment (or nucleotide analog segment, e.g., polynucleotide as defined herein) which can be used to identify a specific polynucleotide sequence present in samples, said nucleic acid segment comprising a nucleotide sequence complementary of the specific polynucleotide sequence to be identified.

The terms “detectable trait” “trait” and “phenotype” are used interchangeably herein and refer to any visible, detectable or otherwise measurable property of an organism such as symptoms of, or susceptibility to a disease for example. Typically the terms “detectable trait” “trait” or “phenotype” are used herein to refer to symptoms of, or susceptibility to a disease; or to refer to an individual's response to an agent, drug, or treatment acting on a disease; or to refer to symptoms of, or susceptibility to side effects to an agent acting on a disease.

The term “treatment” is used herein to encompass any medical intervention known in the art including, for example, the administration of pharmaceutical agents, medically prescribed changes in diet, or habits such as a reduction in smoking or drinking, surgery, the application of medical devices, and the application or reduction of certain physical conditions, for example, light or radiation.

The term “allele” is used herein to refer to variants of a nucleotide sequence. A biallelic polymorphism has two forms; designated herein as the 1^(ST) allele and the 2^(ND) allele. Diploid organisms may be homozygous or heterozygous for an allelic form.

The term “heterozygosity rate” is used herein to refer to the incidence of individuals in a population, which are heterozygous at a particular allele. In a biallelic system the heterozygosity rate is on average equal to 2P_(a)(1−P_(a)), where P_(a) is the frequency of the least common allele. In order to be useful in genetic studies a genetic marker should have an adequate level of heterozygosity to allow a reasonable probability that a randomly selected person will be heterozygous.

The term “genotype” as used herein refers the identity of the alleles present in an individual or a sample. In the context of the present invention a genotype preferably refers to the description of the biallelic marker alleles present in an individual or a sample. The term “genotyping” a sample or an individual for a biallelic marker consists of determining the specific allele or the specific nucleotide carried by an individual at a biallelic marker.

The term “mutation” as used herein refers to a difference in DNA sequence between or among different genomes or individuals which has a frequency below 1%.

The term “haplotype” refers to a combination of alleles present in an individual or a sample. In the context of the present invention a haplotype preferably refers to a combination of biallelic marker alleles found in a given individual and which may be associated with a phenotype.

The term “polymorphism” as used herein refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. “Polymorphic” refers to the condition in which two or more variants of a specific genomic sequence can be found in a population. A “polymorphic site” is the locus at which the variation occurs. A single nucleotide polymorphism is a single base pair change. Typically a single nucleotide polymorphism is the replacement of one nucleotide by another nucleotide at the polymorphic site. Deletion of a single nucleotide or insertion of a single nucleotide, also give rise to single nucleotide polymorphisms. In the context of the present invention “single nucleotide polymorphism” preferably refers to a single nucleotide substitution. Typically, between different genomes or between different individuals, the polymorphic site may be occupied by two different nucleotides.

The terms “biallelic polymorphism” and “biallelic marker” are used interchangeably herein to refer to a polymorphism having two alleles at a fairly high frequency in the population, preferably a single nucleotide polymorphism. A “biallelic marker allele” refers to the nucleotide variants present at a biallelic marker site. Typically the frequency of the less common allele of the biallelic markers of the present invention has been validated to be greater than 1%, preferably the frequency is greater than 10%, more preferably the frequency is at least 20% (i.e. heterozygosity rate of at least 0.32), even more preferably the frequency is at least 30% (i.e. heterozygosity rate of at least 0.42). A biallelic marker wherein the frequency of the less common allele is 30% or more is termed a “high quality biallelic marker.”

The location of nucleotides in a polynucleotide with respect to the center of the polynucleotide are described herein in the following manner. When a polynucleotide has an odd number of nucleotides, the nucleotide at an equal distance from the 3′ and 5′ ends of the polynucleotide is considered to be “at the center” of the polynucleotide, and any nucleotide immediately adjacent to the nucleotide at the center, or the nucleotide at the center itself is considered to be “within 1 nucleotide of the center.” With an odd number of nucleotides in a polynucleotide any of the five nucleotides positions in the middle of the polynucleotide would be considered to be within 2 nucleotides of the center, and so on. When a polynucleotide has an even number of nucleotides, there would be a bond and not a nucleotide at the center of the polynucleotide. Thus, either of the two central nucleotides would be considered to be “within 1 nucleotide of the center” and any of the four nucleotides in the middle of the polynucleotide would be considered to be “within 2 nucleotides of the center”, and so on. For polymorphisms which involve the substitution, insertion or deletion of 1 or more nucleotides, the polymorphism, allele or biallelic marker is “at the center” of a polynucleotide if the difference between the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 3′ end of the polynucleotide, and the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 5′ end of the polynucleotide is zero or one nucleotide. If this difference is 0 to 3, then the polymorphism is considered to be “within 1 nucleotide of the center.” If the difference is 0 to 5, the polymorphism is considered to be “within 2 nucleotides of the center.” If the difference is 0 to 7, the polymorphism is considered to be “within 3 nucleotides of the center,” and so on. For polymorphisms which involve the substitution, insertion or deletion of 1 or more nucleotides, the polymorphism, allele or biallelic marker is “at the center” of a polynucleotide if the difference between the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 3′ end of the polynucleotide, and the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 5′ end of the polynucleotide is zero or one nucleotide. If this difference is 0 to 3, then the polymorphism is considered to be “within 1 nucleotide of the center.” If the difference is 0 to 5, the polymorphism is considered to be “within 2 nucleotides of the center.” If the difference is 0 to 7, the polymorphism is considered to be “within 3 nucleotides of the center,” and so on.

The term “upstream” is used herein to refer to a location which, is toward the 5′ end of the polynucleotide from a specific reference point.

The terms “base paired” and “Watson & Crick base paired” are used interchangeably herein to refer to nucleotides which can be hydrogen bonded to one another be virtue of their sequence identities in a manner like that found in double-helical DNA with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds (See Stryer, L., Biochemistry, 4th edition, 1995).

The terms “complementary” or “complement thereof” are used herein to refer to the sequences of polynucleotides which is capable of forming Watson & Crick base pairing with another specified polynucleotide throughout the entirety of the complementary region. This term is applied to pairs of polynucleotides based solely upon their sequences and not any particular set of conditions under which the two polynucleotides would actually bind.

As used herein the term “map-related biallelic marker” relates to a biallelic marker in linkage disequilibrium with any of the sequences disclosed in SEQ ID Nos. 1 to 3908 which contain a biallelic marker of the map. The term map-related biallelic marker encompasses all of the biallelic markers disclosed in SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908. The preferred map-related biallelic marker alleles of the present invention include each one of the alleles selected individually or in any combination from the biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908, as identified in field <223> of the allele feature in the appended Sequence Listing, individually or in groups consisting of all the possible combinations of the alleles.

The terms “1^(ST) allele” and “2^(ND) allele” refer to the nucleotide located at the polymorphic base of a polynucleotide sequence containing a biallelic marker, as identified in field <222> of the allele feature in the appended Sequence Listing for each Sequence ID number. As used herein, the polymorphic base is located at nucleotide position 24 for each of SEQ ID Nos. 1 to 3908, with the exception of SEQ ID Nos. 914, 1013, 2544, 3434, 3795, and 3028. The polymorphic base is located at nucleotide position 23 for SEQ ID Nos. 914, 1013 and 2544, at nucleotide position 21 for SEQ ID No. 3028, at nucleotide position 20 for SEQ ID No. 3434.

I. Biallelic Markers And Polynucleotides Comprising Biallelic Markers Polynucleotides of the Present Invention

The present invention encompasses polynucleotides for use as primers and probes in the methods of the invention. All of the polynucleotides of the invention may be specified as being isolated, purified or recombinant. These polynucleotides may consist of, consist essentially of, or comprise a contiguous span of nucleotides of a sequence from any sequence in the Sequence Listing as well as sequences which are complementary thereto (“complements thereof”). The “contiguous span” may be at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides in length, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID. It should be noted that the polynucleotides of the present invention are not limited to having the exact flanking sequences surrounding the polymorphic bases which are enumerated in the Sequence Listing. Rather, it will be appreciated that the flanking sequences surrounding the biallelic markers, or any of the primers of probes of the invention which, are more distant from the markers, may be lengthened or shortened to any extent compatible with their intended use and the present invention specifically contemplates such sequences. It will be appreciated that the polynucleotides referred to in the Sequence Listing may be of any length compatible with their intended use. Also the flanking regions outside of the contiguous span need not be homologous to native flanking sequences which actually occur in human subjects. The addition of any nucleotide sequence, which is compatible with the nucleotides intended use is specifically contemplated. The contiguous span may optionally include the map-related biallelic marker in said sequence. Biallelic markers generally consist of a polymorphism at one single base position. Each biallelic marker therefore corresponds to two forms of a polynucleotide sequence which, when compared with one another, present a nucleotide modification at one position. Usually, the nucleotide modification involves the substitution of one nucleotide for another. Optionally either the 1^(ST) allele or the 2^(ND) allele of the biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908 may be specified as being present at the map-related biallelic marker.

Preferred polynucleotides may consist of, consist essentially of, or comprise a contiguous span of nucleotides of a sequence from SEQ ID Nos. 1 to 2260 as well as sequences which are complementary thereto. The “contiguous span” may be at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides in length, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID. Particularly preferred are polynucleotides which consist of, consist essentially of, or comprise a contiguous span of nucleotides of a sequence of any of SEQ ID Nos. 1 to 2260, or the complements thereof, wherein the 1^(ST) allele of the biallelic marker of the SEQ ID No. is present at the map-related biallelic marker. Other preferred polynucleotides consist of, consist essentially of, or comprise a contiguous span of nucleotides of any of SEQ ID Nos. 1 to 2260, or the complements thereof, wherein the 2^(ND) allele of the biallelic marker of the SEQ ID No. is present at the map-related biallelic marker. Preferred polynucleotides may consist of, consist essentially of, or comprise a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides in length, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID No., of a sequence from SEQ ID Nos. 2261 to 3734 as well as sequences which are complementary thereto. Particularly preferred are polynucleotides which consist of, consist essentially of, or comprise a contiguous span of nucleotides of a sequence of any of SEQ ID Nos. 2261 to 3734, or the complements thereof, wherein the 1^(ST) allele of the biallelic marker of the SEQ ID No. is present at the map-related biallelic marker. Other preferred polynucleotides consist of, consist essentially of, or comprise a contiguous span of nucleotides of any of SEQ ID Nos. 2261 to 3734, or the complements thereof, wherein the 2^(ND) allele of the biallelic marker of the SEQ ID No. is present at the map-related biallelic marker. Preferred polynucleotides may consist of, consist essentially of, or comprise a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides in length, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID No., of a sequence from SEQ ID Nos. 3735 to 3908 as well as sequences which are complementary thereto. Particularly preferred are polynucleotides which consist of, consist essentially of, or comprise a contiguous span of nucleotides of a sequence of any of SEQ ID Nos. 3735 to 3908, or the complements thereof, wherein the 1^(ST) allele of the biallelic marker of the SEQ ID No. is present at the map-related biallelic marker. Other preferred polynucleotides consist of, consist essentially of, or comprise a contiguous span of nucleotides of any of SEQ ID Nos. 3735 to 3908, or the complements thereof, wherein the 2^(ND) allele of the biallelic marker of the SEQ ID No. is present at the map-related biallelic marker. Also encompassed by the polynucleotides of the present invention are polynucleotides which consist of, consist essentially of, or comprise a contiguous span at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides in length, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of a sequence from SEQ ID Nos. 1201, 3242, 3907 and 3908 as well as sequences which are complementary thereto, wherein said contiguous span of SEQ ID Nos. 1201 or 3242 contains a “G” at the polymorphic base, or wherein said contiguous span of SEQ ID Nos. 3907 or 3908 contain an “A” at the polymorphic base.

The present invention also relates to a biallelic marker or set of biallelic markers of the invention comprising:

(a) at least one of SEQ ID Nos. 583, 620, 1277 to 1279, 1281, 1375 to 1377, 1379 to 1382, 1676 to 1681, 3106, 3547, 3548, 3889; and/or

(b) at least one of SEQ ID Nos. 86, 105, 109, 110, 185, 284, 381, 414, 428, 441, 445, 446, 453, 464, 467, 487, 489, 520, 3915 to 3918, 3920, and 3923 to 3926; and/or

(c) at least one of SEQ ID Nos. 232 to 237, 340, 346, and 3927-3934; and/or

(d) at least one of SEQ ID Nos. 607, 616, 619, 623, 626, 627, 645, 646, 650, 651, 1899 and 2721; and/or

(e) at least one of SEQ ID Nos. 2694 to 2697, 3494 to 3496 and 3882; and/or

(f) at least one of SEQ ID Nos. 204, 205, 225, 273, 274, 1723, 1732, 1743.

Thus, in said embodiment, the polynucleotides and nucleic acid codes of the invention may comprise a nucleotide sequence or group of nucleotide sequences of said SEQ ID numbers listed above in (a) to (f), the amplification primers related to said SEQ ID Numbers, as described in Table 1, and the sequences complementary thereto. Optionally, any biallelic markers, sets of biallelic markers, polynucleotides or nucleic acid codes described throughout the present specification may be selected from a group specifically excluding one or more of said SEQ ID numbers listed above in (a) to (f). The biallelic markers, sets of biallelic markers, polynucleotides or nucleic acid codes of the invention may be selected from a group which specifically excludes one or more of said SEQ ID numbers listed above in (a) to (f) individually or in any combination.

The invention also relates to polynucleotides that hybridize, under conditions of high or intermediate stringency, to a polynucleotide of a sequence from any of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908 as well as sequences which are complementary thereto. Preferably such polynucleotides are at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides in length, to the extent that a polynucleotide of these lengths is consistent with the lengths of the particular Sequence ID. Preferred polynucleotides comprise a map-related biallelic marker. Optionally either the 1^(ST) or the 2^(ND) allele of the biallelic markers disclosed in the SEQ ID No. may be specified as being present at the map-related biallelic marker. Conditions of high and intermediate stringency are further described in III.C.4.

The primers of the present invention may be designed from the disclosed sequences using any method known in the art. A preferred set of primers is fashioned such that the 3′ end of the contiguous span of identity with the sequences of the Sequence Listing is present at the 3′ end of the primer. Such a configuration allows the 3′ end of the primer to hybridize to a selected nucleic acid sequence and dramatically increases the efficiency of the primer for amplification or sequencing reactions.

In a preferred set of primers, the contiguous span is found in one of the sequences described in SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 or the complements thereof. The invention also relates to polynucleotides consisting of, consisting essentially of, or comprising a contiguous span of nucleotides of a sequence from SEQ ID Nos. 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773, as well as sequences which are complementary thereto, wherein the “contiguous span” may be at least 8, 10, 12, 15, 18, 19, 20, or 21 nucleotides in length, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID No.

Allele specific primers may be designed such that a biallelic marker is at the 3′ end of the contiguous span and the contiguous span is present at the 3′ end of the primer. Such allele specific primers tend to selectively prime an amplification or sequencing reaction so long as they are used with a nucleic acid sample that contains one of the two alleles present at a biallelic marker. The 3′ end of primer of the invention may be located within or at least 2, 4, 6, 8, 10, to the extent that this distance is consistent with the particular Sequence ID, nucleotides upstream of a map-related biallelic marker in said sequence or at any other location which is appropriate for their intended use in sequencing, amplification or the location of novel sequences or markers. Primers with their 3′ ends located 1 nucleotide upstream of a map-related biallelic marker have a special utility as microsequencing assays. Preferred microsequencing primers are described in SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908, where for each of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908, the sense microsequencing primer contains the complement of the 19 nucleotides having their 3′ ends located 1 nucleotide upstream of the polymorphic base of the respective SEQ ID No, and where the antisense microsequencing primer contains the complement of the 19 nucleotides of the complementary strand, nucleotides of the primer having their 3′ end located 1 nucleotide upstream of the polymorphic base on the complementary strand to the respective SEQ ID No. The most preferred of said microsequencing primers for each of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908 are microsequencing primers indicated as “A” or “S” in Table 1, which have been validated in microsequencing experiments.

The probes of the present invention may be designed from the disclosed sequences for any method known in the art, particularly methods which allow for testing if a particular sequence or marker disclosed herein is present. A preferred set of probes may be designed for use in the hybridization assays of the invention in any manner known in the art such that they selectively bind to one allele of a biallelic marker, but not the other under any particular set of assay conditions. Preferred hybridization probes may consist of, consist essentially of, or comprise a contiguous span of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908, or the complement thereof, which ranges in length from least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID No., or be specified as being 12, 15, 18, 19, 20, 25, 35, 40, 43, 44, 45, 46 or 47 nucleotides in length and including the map-related biallelic marker of said sequence. Optionally the 1 st allele or 2nd allele of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908 may be specified as being present at the biallelic marker site. Optionally, said biallelic marker may be within 6, 5, 4, 3, 2, or 1 nucleotides of the center of the hybridization probe or at the center of said probe.

Any of the polynucleotides of the present invention can be labeled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive substances, fluorescent dyes or biotin. Preferably, polynucleotides are labeled at their 3′ and 5′ ends. A label can also be used to capture the primer, so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support. A capture label is attached to the primers or probes and can be a specific binding member which forms a binding pair with the solid's phase reagent's specific binding member (e.g. biotin and streptavidin). Therefore depending upon the type of label carried by a polynucleotide or a probe, it may be employed to capture or to detect the target DNA. Further, it will be understood that the polynucleotides, primers or probes provided herein, may, themselves, serve as the capture label. For example, in the case where a solid phase reagent's binding member is a nucleic acid sequence, it may be selected such that it binds a complementary portion of a primer or probe to thereby immobilize the primer or probe to the solid phase. In cases where a polynucleotide probe itself serves as the binding member, those skilled in the art will recognize that the probe will contain a sequence or “tail” that is not complementary to the target. In the case where a polynucleotide primer itself serves as the capture label, at least a portion of the primer will be free to hybridize with a nucleic acid on a solid phase. DNA Labeling techniques are well known to the skilled technician.

Any of the polynucleotides, primers and probes of the present invention can be conveniently immobilized on a solid support. Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes® and others. The solid support is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtiter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples. Suitable methods for immobilizing nucleic acids on solid phases include ionic, hydrophobic, covalent interactions and the like. A solid support, as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent. The additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent. As yet another alternative, the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid support and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid support material before the performance of the assay or during the performance of the assay. The solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes® and other configurations known to those of ordinary skill in the art. The polynucleotides of the invention can be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the inventions to a single solid support. In addition, polynucleotides other than those of the invention may attached to the same solid support as one or more polynucleotides of the invention.

Any polynucleotide provided herein may be attached in overlapping areas or at random locations on the solid support. Alternatively the polynucleotides of the invention may be attached in an ordered array wherein each polynucleotide is attached to a distinct region of the solid support which does not overlap with the attachment site of any other polynucleotide. Preferably, such an ordered array of polynucleotides is designed to be “addressable” where the distinct locations are recorded and can be accessed as part of an assay procedure. Addressable polynucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. The knowledge of the precise location of each polynucleotides location makes these “addressable” arrays particularly useful in hybridization assays. Any addressable array technology known in the art can be employed with the polynucleotides of the invention. One particular embodiment of these polynucleotide arrays is known as the Genechips™, and has been generally described in U.S. Pat. No. 5,143,854; PCT publications WO 90/15070 and 92/10092, the disclosures of which are incorporated herein by reference in their entireties. These arrays may generally be produced using mechanical synthesis methods or light directed synthesis methods, which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis (Fodor et al., Science, 251:767-777, 1991, the disclosure of which is incorporated herein by reference in its entirety). The immobilization of arrays of oligonucleotides on solid supports has been rendered possible by the development of a technology generally identified as “Very Large Scale Immobilized Polymer Synthesis” (VLSIPS™) in which, typically, probes are immobilized in a high density array on a solid surface of a chip. Examples of VLSIPS™ technologies are provided in U.S. Pat. Nos. 5,143,854 and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092 and WO 95/11995, the disclosures of which are incorporated herein by reference in their entirety, which describe methods for forming oligonucleotide arrays through techniques such as light-directed synthesis techniques. In designing strategies aimed at providing arrays of nucleotides immobilized on solid supports, further presentation strategies were developed to order and display the oligonucleotide arrays on the chips in an attempt to maximize hybridization patterns and sequence information. Examples of such presentation strategies are disclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256, the disclosures of which are incorporated herein by reference in their entireties.

Oligonucleotide arrays may comprise at least one of the sequences selected from the group consisting of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908 and the sequences complementary thereto, or a fragment thereof of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 consecutive nucleotides, to the extent that fragments of these lengths is consistent with the lengths of the particular Sequence ID, for determining whether a sample contains one or more alleles of the biallelic markers of the present invention. Oligonucleotide arrays may also comprise at least one of the sequences selected from the group consisting of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908, and the sequences complementary thereto, or a fragment thereof of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 consecutive nucleotides, to the extent that fragments of these lengths is consistent with the lengths of the particular Sequence ID, for amplifying one or more alleles of the biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908. In other embodiments, arrays may also comprise at least one of the sequences selected from the group consisting of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908 and the sequences complementary thereto, or a fragment thereof of at 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 consecutive nucleotides, to the extent that fragments of these lengths is consistent with the lengths of the particular Sequence ID, for conducting microsequencing analyses to determine whether a sample contains one or more alleles of the biallelic markers of the invention. In still further embodiments, the oligonucleotide array may comprise at least one of the sequences selected from the group consisting of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908 and the sequences complementary thereto, or a fragment thereof of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides in length, to the extent that fragments of these lengths is consistent with the lengths of the particular Sequence ID, for determining whether a sample contains one or more alleles of the biallelic markers of the present invention.

In designing strategies aimed at providing arrays of nucleotides immobilized on solid supports, further presentation strategies were developed to order and display the probe arrays on the chips in an attempt to maximize hybridization patterns and sequence information. Examples of such presentation strategies are disclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256, the disclosures of which are incorporated herein by reference in their entireties.

Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged in a grid-like pattern and miniaturized to the size of a dime. In some embodiments, the efficiency of hybridization of nucleic acids in the sample with the probes attached to the chip may be improved by using polyacrylamide gel pads isolated from one another by hydrophobic regions in which the DNA probes are covalently linked to an acrylamide matrix.

The polymorphic bases present in the biallelic marker or markers of the sample nucleic acids are determined as follows. Probes which contain at least a portion of one or more of the biallelic markers of the present invention are synthesized either in situ or by conventional synthesis and immobilized on an appropriate chip using methods known to the skilled technician.

Any one or more alleles of the biallelic markers described herein (SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto) or fragments thereof containing the polymorphic bases, may be fixed to a solid support, such as a microchip or other immobilizing surface. The fragments of these nucleic acids may comprise at least 10, at least 15, at least 20, at least 25, or more than 25 consecutive nucleotides of the biallelic markers described herein. Preferably, the fragments include the polymorphic bases of the biallelic markers.

A nucleic acid sample is applied to the immobilizing surface and analyzed to determine the identities of the polymorphic bases of one or more of the biallelic markers. In some embodiments, the solid support may also include one or more of the amplification primers described herein, or fragments comprising at least 10, at least 15, or at least 20 consecutive nucleotides thereof, for generating an amplification product containing the polymorphic bases of the biallelic markers to be analyzed in the sample.

Another embodiment of the present invention is a solid support which includes one or more of the microsequencing primers of the invention, or fragments comprising at least 10, at least 15, or at least 20 consecutive nucleotides thereof and having a 3′ terminus immediately upstream of the polymorphic base of the corresponding biallelic marker, for determining the identity of the polymorphic base of the one or more biallelic markers fixed to the solid support.

For example, one embodiment of the present invention is an array of nucleic acids fixed to a solid support, such as a microchip, bead, or other immobilizing surface, comprising one or more of the biallelic markers in the maps of the present invention or a fragment comprising at least 10, at least 15, at least 20, at least 25, or more than 25 consecutive nucleotides thereof including the polymorphic base. For example, the array may comprise 1, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, or 3000 of the biallelic markers selected from the group consisting of SEQ ID Nos.: 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto, or a fragment comprising at least 10, at least 15, at least 20, at least 25, or more than 25 consecutive nucleotides thereof including the polymorphic base.

Another embodiment of the present invention is an array comprising amplification primers for generating amplification products containing the polymorphic bases of one or more, at least five, at least 10, at least 20, at least 100, at least 200, at least 300, at least 400, or more than 400 of the biallelic markers in the maps of the present invention. For example, the array may comprise amplification primers for generating amplification products containing the polymorphic bases of at least 1,5, 10, 20, 50, 100, 200, 300, 400, 500, 1000, 2000, or 3000, of the biallelic markers selected from the group consisting of SEQ ID Nos.: 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto. In such arrays, the amplification primers included in the array are capable of amplifying the biallelic marker sequences to be detected in the nucleic acid sample applied to the array (i.e. the amplification primers correspond to the biallelic markers affixed to the array—see Table 1). Thus, the arrays may include one or more of the amplification primers of SEQ ID Nos.: 3935 to 7842, 7866 to 11773, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 10125, 10126 to 11599, and 11600 to 11773 corresponding to the one or more biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908 which are included in the array.

Another embodiment of the present invention is an array which includes microsequencing primers capable of determining the identity of the polymorphic bases of at least 1, 5, 10, 20, 50, 100, 200, 300, 500, 1000, 2000, or 3000 of the present invention. For example, the array may comprise microsequencing primers capable of determining the identity of the polymorphic bases of one or more, at least five, at least 10, at least 20, at least 100, at least 200, at least 300, at least 400, or more than 400 of the biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto.

Arrays containing any combination of the above nucleic acids which permits the specific detection or identification of the polymorphic bases of the biallelic markers in the maps of the present invention, including any combination of biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto are also within the scope of the present invention. For example, the array may comprise both the biallelic markers and amplification primers capable of generating amplification products containing the polymorphic bases of the biallelic markers. Alternatively, the array may comprise both amplification primers capable of generating amplification products containing the polymorphic bases of the biallelic markers and microsequencing primers capable of determining the identities of the polymorphic bases of these markers.

Although the above examples describe arrays comprising specific groups of biallelic markers and, in some embodiments, specific amplification primers and microsequencing primers, it will be appreciated that the present invention encompasses arrays including any biallelic marker, group of biallelic markers, amplification primer, group of amplification primers, microsequencing primer, or group of amplification primers described herein, as well as any combination of the preceding nucleic acids.

The present invention also encompasses diagnostic kits comprising one or more polynucleotides of the invention, optionally with a portion or all of the necessary reagents and instructions for genotyping a test subject by determining the identity of a nucleotide at a map-related biallelic marker. The polynucleotides of a kit may optionally be attached to a solid support, or be part of an array or addressable array of polynucleotides. The kit may provide for the determination of the identity of the nucleotide at a marker position by any method known in the art including, but not limited to, a sequencing assay method, a microsequencing assay method, a hybridization assay method, or an allele specific amplification method. Optionally,such a kit may include instructions for scoring the results of the determination with respect to the test subjects′ risk of contracting a diseases involving a disease, likely response to an agent acting on a disease, or chances of suffering from side effects to an agent acting on a disease.

II. Methods For De Novo Identification Of Biallelic Markers

Any of a variety of methods can be used to screen a genomic fragment for single nucleotide polymorphisms such as differential hybridization with oligonucleotide probes, detection of changes in the mobility measured by gel electrophoresis or direct sequencing of the amplified nucleic acid. A preferred method for identifying biallelic markers involves comparative sequencing of genomic DNA fragments from an appropriate number of unrelated individuals.

In a first embodiment, DNA samples from unrelated individuals are pooled together, following which the genomic DNA of interest is amplified and sequenced. The nucleotide sequences thus obtained are then analyzed to identify significant polymorphisms. One of the major advantages of this method resides in the fact that the pooling of the DNA samples substantially reduces the number of DNA amplification reactions and sequencing reactions, which must be carried out. Moreover, this method is sufficiently sensitive so that a biallelic marker obtained thereby usually demonstrates a sufficient frequency of its less common allele to be useful in conducting association studies. Usually, the frequency of the least common allele of a biallelic marker identified by this method is at least 10%.

In a second embodiment, the DNA samples are not pooled and are therefore amplified and sequenced individually. This method is usually preferred when biallelic markers need to be identified in order to perform association studies within candidate genes. Preferably, highly relevant gene regions such as promoter regions or exon regions may be screened for biallelic markers. A biallelic marker obtained using this method may show a lower degree of informativeness for conducting association studies, e.g. if the frequency of its less frequent allele may be less than about 10%. Such a biallelic marker will however be sufficiently informative to conduct association studies and it will further be appreciated that including less informative biallelic markers in the genetic analysis studies of the present invention, may allow in some cases the direct identification of causal mutations, which may, depending on their penetrance, be rare mutations.

The following is a description of the various parameters of a preferred method used by the inventors for the identification of the biallelic markers of the present invention.

II.A. Genomic DNA Samples

The genomic DNA samples from which the biallelic markers of the present invention are generated are preferably obtained from unrelated individuals corresponding to a heterogeneous population of known ethnic background. The number of individuals from whom DNA samples are obtained can vary substantially, preferably from about 10 to about 1000, more preferably from about 50 to about 200 individuals. Usually, DNA samples are collected from at least about 100 individuals in order to have sufficient polymorphic diversity in a given population to identify as many markers as possible and to generate statistically significant results.

As for the source of the genomic DNA to be subjected to analysis, any test sample can be foreseen without any particular limitation. These test samples include biological samples, which can be tested by the methods of the present invention described herein, and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supernatants; fixed tissue specimens including tumor and non-tumor tissue and lymph node tissues; bone marrow aspirates and fixed cell specimens. The preferred source of genomic DNA used in the present invention is from peripheral venous blood of each donor. Techniques to prepare genomic DNA from biological samples are well known to the skilled technician. Details of a preferred embodiment are provided in Example 27. The person skilled in the art can choose to amplify pooled or unpooled DNA samples.

II.B. DNA Amplification

The identification of biallelic markers in a sample of genomic DNA may be facilitated through the use of DNA amplification methods. DNA samples can be pooled or unpooled for the amplification step. DNA amplification techniques are well known to those skilled in the art. Various methods to amplify DNA fragments carrying biallelic markers are further described hereinafter in III.B. The PCR technology is the preferred amplification technique used to identify new biallelic markers.

In a first embodiment, biallelic markers are identified using genomic sequence information generated by the inventors. Genomic DNA fragments, such as the inserts of the BAC clones described above, are sequenced and used to design primers for the amplification of 500 bp fragments. These 500 bp fragments are amplified from genomic DNA and are scanned for biallelic markers. Primers may be designed using the OSP software (Hillier L. and Green P., 1991, the entire disclosure of which is incorporated by reference herein in its entirety). All primers may contain, upstream of the specific target bases, a common oligonucleotide tail that serves as a sequencing primer. Those skilled in the art are familiar with primer extensions, which can be used for these purposes.

In another embodiment of the invention, genomic sequences of candidate genes are available in public databases allowing direct screening for biallelic markers. Preferred primers, useful for the amplification of genomic sequences encoding the candidate genes, focus on promoters, exons and splice sites of the genes. A biallelic marker present in these functional regions of the gene have a higher probability to be a causal mutation.

Preferred primers include those disclosed in SEQ ID Nos. 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773.

II.C. Sequencing Of Amplified Genomic DNA And Identification Of Single Nucleotide Polymorphisms

The amplification products generated as described above, are then sequenced using any method known and available to the skilled technician. Methods for sequencing DNA using either the dideoxy-mediated method (Sanger method) or the Maxam-Gilbert method are widely known to those of ordinary skill in the art. Such methods are for example disclosed in Maniatis et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Second Edition, 1989 the disclosure of which is incorporated herein by reference in its entirety). Alternative approaches include hybridization to high-density DNA probe arrays as described in Chee et al. (Science 274, 610, 1996, the disclosure of which is incorporated herein by reference in its entirety).

Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. The products of the sequencing reactions are run on sequencing gels and the sequences are determined using gel image analysis. The polymorphism search is based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position. Because each dideoxy terminator is labeled with a different fluorescent molecule, the two peaks corresponding to a biallelic site present distinct colors corresponding to two different nucleotides at the same position on the sequence. However, the presence of two peaks can be an artifact due to background noise. To exclude such an artifact, the two DNA strands are sequenced and a comparison between the peaks is carried out. In order to be registered as a polymorphic sequence, the polymorphism has to be detected on both strands.

The above procedure permits those amplification products, which contain biallelic markers to be identified. The detection limit for the frequency of biallelic polymorphisms detected by sequencing pools of 100 individuals is approximately 0.1 for the minor allele, as verified by sequencing pools of known allelic frequencies. However, more than 90% of the biallelic polymorphisms detected by the pooling method have a frequency for the minor allele higher than 0.25. Therefore, the biallelic markers selected by this method have a frequency of at least 0.1 for the minor allele and less than 0.9 for the major allele. Preferably at least 0.2 for the minor allele and less than 0.8 for the major allele, more preferably at least 0.3 for the minor allele and less than 0.7 for the major allele, thus a heterozygosity rate higher than 0.18, preferably higher than 0.32, more preferably higher than 0.42.

In another embodiment, biallelic markers are detected by sequencing individual DNA samples, the frequency of the minor allele of such a biallelic marker may be less than 0.1.

The markers carried by the same fragment of genomic DNA, such as the insert in a BAC clone, need not necessarily be ordered with respect to one another within the genomic fragment to conduct association studies. However, in some embodiments of the present invention, the order of biallelic markers carried by the same fragment of genomic DNA are determined.

II.D. Validation of the Biallelic Markers of the Present Invention

The polymorphisms are evaluated for their usefulness as genetic markers by validating that both alleles are present in a population. Validation of the biallelic markers is accomplished by genotyping a group of individuals by a method of the invention and demonstrating that both alleles are present. Microsequencing is a preferred method of genotyping alleles. The validation by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual. The group can be as small as one individual if that individual is heterozygous for the allele in question. Preferably the group contains at least three individuals, more preferably the group contains five or six individuals, so that a single validation test will be more likely to result in the validation of more of the biallelic markers that are being tested. It should be noted, however, that when the validation test is performed on a small group it may result in a false negative result if as a result of sampling error none of the individuals tested carries one of the two alleles. Thus, the validation process is less useful in demonstrating that a particular initial result is an artifact, than it is at demonstrating that there is a bonafide biallelic marker at a particular position in a sequence. All of the genotyping, haplotyping, association, and interaction study methods of the invention may optionally be performed solely with validated biallelic markers.

II.E. Evaluation of the Frequency of the Biallelic Markers of the Present Invention

The validated biallelic markers are further evaluated for their usefulness as genetic markers by determining the frequency of the least common allele at the biallelic marker site. The determination of the least common allele is accomplished by genotyping a group of individuals by a method of the invention and demonstrating that both alleles are present. This determination of frequency by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual. The group must be large enough to be representative of the population as a whole. Preferably the group contains at least 20 individuals, more preferably the group contains at least 50 individuals, most preferably the group contains at least 100 individuals. Of course the larger the group the greater the accuracy of the frequency determination because of reduced sampling error. A biallelic marker wherein the frequency of the less common allele is 30% or more is termed a “high quality biallelic marker.” All of the genotyping, haplotyping, association, and interaction study methods of the invention may optionally be performed solely with high quality biallelic markers.

III. Methods Of Genotyping An Individual For Biallelic Markers

Methods are provided to genotype a biological sample for one or more biallelic markers of the present invention, all of which may be performed in vitro. Such methods of genotyping comprise determining the identity of a nucleotide at a map-related biallelic marker by any method known in the art. These methods find use in genotyping case-control populations in association studies as well as individuals in the context of detection of alleles of biallelic markers which, are known to be associated with a given trait, in which case both copies of the biallelic marker present in individual's genome are determined so that an individual may be classified as homozygous or heterozygous for a particular allele.

These genotyping methods can be performed nucleic acid samples derived from a single individual or pooled DNA samples.

Genotyping can be performed using similar methods as those described above for the identification of the biallelic markers, or using other genotyping methods such as those further described below. In preferred embodiments, the comparison of sequences of amplified genomic fragments from different individuals is used to identify new biallelic markers whereas microsequencing is used for genotyping known biallelic markers in diagnostic and association study applications.

III.A. Source of DNA for Genotyping

Any source of nucleic acids, in purified or non-purified form, can be utilized as the starting nucleic acid, provided it contains or is suspected of containing the specific nucleic acid sequence desired. DNA or RNA may be extracted from cells, tissues, body fluids and the like as described above in II.A. While nucleic acids for use in the genotyping methods of the invention can be derived from any mammalian source, the test subjects and individuals from which nucleic acid samples are taken are generally understood to be human.

III.B. Amplification Of DNA Fragments Comprising Biallelic Markers

Methods and polynucleotides are provided to amplify a segment of nucleotides comprising one or more biallelic marker of the present invention. It will be appreciated that amplification of DNA fragments comprising biallelic markers may be used in various methods and for various purposes and is not restricted to genotyping. Nevertheless, many genotyping methods, although not all, require the previous amplification of the DNA region carrying the biallelic marker of interest. Such methods specifically increase the concentration or total number of sequences that span the biallelic marker or include that site and sequences located either distal or proximal to it. Diagnostic assays may also rely on amplification of DNA segments carrying a biallelic marker of the present invention.

Amplification of DNA may be achieved by any method known in the art. The established PCR (polymerase chain reaction) method or by developments thereof or alternatives. Amplification methods which can be utilized herein include but are not limited to Ligase Chain Reaction (LCR) as described in EP A 320 308 and EP A 439 182, Gap LCR (Wolcott, M. J., Clin. Mcrobiol. Rev. 5:370-386), the so-called “NASBA” or “3SR” technique described in Guatelli J. C. et al. (Proc. Natl. Acad. Sci. USA 87:1874-1878, 1990) and in Compton J. (Nature 350:91-92, 1991), Q-beta amplification as described in European Patent Application no 4544610, strand displacement amplification as described in Walker et al. (Clin. Chem. 42:9-13, 1996) and EP A 684 315 and, target mediated amplification as described in PCT Publication WO 9322461, the disclosures of which are incorporated herein by reference in their entireties.

LCR and Gap LCR are exponential amplification techniques, both depend on DNA ligase to join adjacent primers annealed to a DNA molecule. In Ligase Chain Reaction (LCR), probe pairs are used which include two primary (first and second) and two secondary (third and fourth) probes, all of which are employed in molar excess to target. The first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5′ phosphate-3′hydroxyl relationship, and so that a ligase can covalently fuse or ligate the two probes into a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting fashion. Of course, if the target is initially double stranded, the secondary probes also will hybridize to the target complement in the first instance. Once the ligated strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary ligated product. It is important to realize that the ligated products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. A method for multiplex LCR has also been described (WO 9320227, the disclosure of which is incorporated herein by reference in its entirety). Gap LCR (GLCR) is a version of LCR where the probes are not adjacent but are separated by 2 to 3 bases.

For amplification of mRNAs, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770, the disclosure of which is incorporated herein by reference in its entirety, or, to use Asymmetric Gap LCR (RT-AGLCR) as described by Marshall R. L. et al. (PCR Methods and Applications 4:80-84, 1994, the disclosure of which is incorporated herein by reference in its entirety). AGLCR is a modification of GLCR that allows the amplification of RNA.

Some of these amplification methods are particularly suited for the detection of single nucleotide polymorphisms and allow the simultaneous amplification of a target sequence and the identification of the polymorphic nucleotide as it is further described in III.C.

The PCR technology is the preferred amplification technique used in the present invention. A variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see Molecular Cloning to Genetic Engineering White, B.A. Ed. in Methods in Molecular Biology 67: Humana Press, Totowa (1997) and the publication entitled “PCR Methods and Applications” (1991, Cold Spring Harbor Laboratory Press, the disclosures of which are incorporated herein by reference in their entireties). In each of these PCR procedures, PCR primers on either side of the nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase. The nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample. The hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites. PCR has further been described in several patents including U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188, the disclosures of which are incorporated herein by reference in their entireties.

The identification of biallelic markers as described above allows the design of appropriate oligonucleotides, which can be used as primers to amplify DNA fragments comprising the biallelic markers of the present invention. Amplification can be performed using the primers initially used to discover new biallelic markers which are described herein or any set of primers allowing the amplification of a DNA fragment comprising a biallelic marker of the present invention. Primers can be prepared by any suitable method. As for example, direct chemical synthesis by a method such as the phosphodiester method of Narang S. A. et al. (Methods Enzymol. 68:90-98, 1979), the phosphodiester method of Brown E. L. et al. (Methods Enzymol. 68:109-151, 1979), the diethylphosphoramidite method of Beaucage et al. (Tetrahedron Lett. 22:1859-1862, 1981) and the solid support method described in EP 0 707 592, the disclosures of which are incorporated herein by reference in their entireties.

In some embodiments the present invention provides primers for amplifying a DNA fragment containing one or more biallelic markers of the present invention. Preferred amplification primers are listed in SEQ ID Nos. 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773. It will be appreciated that the primers listed are merely exemplary and that any other set of primers which produce amplification products containing one or more biallelic markers of the present invention.

The primers are selected to be substantially complementary to the different strands of each specific sequence to be amplified. The length of the primers of the present invention can range from 8 to 100 nucleotides, preferably from 8 to 50, 8 to 30 or more preferably 8 to 25 nucleotides. Shorter primers tend to lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer primers are expensive to produce and can sometimes self-hybridize to form hairpin structures. The formation of stable hybrids depends on the melting temperature (Tm) of the DNA. The Tm depends on the length of the primer, the ionic strength of the solution and the G+C content. The higher the G+C content of the primer, the higher is the melting temperature because G:C pairs are held by three H bonds whereas A:T pairs have only two. The G+C content of the amplification primers of the present invention preferably ranges between 10 and 75%, more preferably between 35 and 60%, and most preferably between 40 and 55%. The appropriate length for primers under a particular set of assay conditions may be empirically determined by one of skill in the art.

The spacing of the primers determines the length of the segment to be amplified. In the context of the present invention amplified segments carrying biallelic markers can range in size from at least about 25 bp to 35 kbp. Amplification fragments from 25-3000 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100-600 bp are highly preferred. It will be appreciated that amplification primers for the biallelic markers may be any sequence which allow the specific amplification of any DNA fragment carrying the markers. Amplification primers may be labeled or immobilized on a solid support as described in I.

III.C. Methods of Genotyping DNA Samples for Biallelic Markers

Any method known in the art can be used to identify the nucleotide present at a biallelic marker site. Since the biallelic marker allele to be detected has been identified and specified in the present invention, detection will prove simple for one of ordinary skill in the art by employing any of a number of techniques. Many genotyping methods require the previous amplification of the DNA region carrying the biallelic marker of interest. While the amplification of target or signal is often preferred at present, ultrasensitive detection methods which do not require amplification are also encompassed by the present genotyping methods. Methods well-known to those skilled in the art that can be used to detect biallelic polymorphisms include methods such as, conventional dot blot analyzes, single strand conformational polymorphism analysis (SSCP) described by Orita et al. (Proc. Natl. Acad. Sci. U.S.A 86:27776-2770, 1989, the disclosure of which is incorporated herein by reference in its entirety), denaturing gradient gel electrophoresis (DGGE), heteroduplex analysis, mismatch cleavage detection, and other conventional techniques as described in Sheffield, V. C. et al. (Proc. Natl. Acad. Sci. USA 49:699-706, 1991), White et al. (Genomics 12:301-306, 1992), Grompe, M. et al. (Proc. Natl. Acad. Sci. USA 86:5855-5892, 1989) and Grompe, M. (Nature Genetics 5:111-117, 1993, the disclosures of which are incorporated herein by reference in their entireties). Another method for determining the identity of the nucleotide present at a particular polymorphic site employs a specialized exonuclease-resistant nucleotide derivative as described in U.S. Pat. No. 4,656,127, the disclosure of which is incorporated herein by reference in its entirety.

Preferred methods involve directly determining the identity of the nucleotide present at a biallelic marker site by sequencing assay, enzyme-based mismatch detection assay, or hybridization assay. The following is a description of some preferred methods. A highly preferred method is the microsequencing technique. The term “sequencing assay” is used herein to refer to polymerase extension of duplex primer/template complexes and includes both traditional sequencing and microsequencing.

1) Sequencing Assays

The nucleotide present at a polymorphic site can be determined by sequencing methods. In a preferred embodiment, DNA samples are subjected to PCR amplification before sequencing as described above. DNA sequencing methods are described in IIC.

Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. Sequence analysis allows the identification of the base present at the biallelic marker site.

2) Microsequencing Assays

In microsequencing methods, a nucleotide at the polymorphic site that is unique to one of the alleles in a target DNA is detected by a single nucleotide primer extension reaction. This method involves appropriate microsequencing primers which, hybridize just upstream of a polymorphic base of interest in the target nucleic acid. A polymerase is used to specifically extend the 3′ end of the primer with one single ddNTP (chain terminator) complementary to the selected nucleotide at the polymorphic site. Next the identity of the incorporated nucleotide is determined in any suitable way.

Typically, microsequencing reactions are carried out using fluorescent ddNTPs and the extended microsequencing primers are analyzed by electrophoresis on ABI 377 sequencing machines to determine the identity of the incorporated nucleotide as described in EP 412 883, the disclosure of which is incorporated herein by reference in its entirety. Alternatively capillary electrophoresis can be used in order to process a higher number of assays simultaneously. An example of a typical microsequencing procedure that can be used in the context of the present invention is provided in Example 8.

Different approaches can be used to detect the nucleotide added to the microsequencing primer. A homogeneous phase detection method based on fluorescence resonance energy transfer has been described by Chen and Kwok (Nucleic Acids Research 25:347-353 1997) and Chen et al. (Proc. Natl. Acad. Sci. USA 94/20 10756-10761,1997, the disclosures of which are incorporated herein by reference in their entireties). In this method, amplified genomic DNA fragments containing polymorphic sites are incubated with a 5′-fluorescein-labeled primer in the presence of allelic dye-labeled dideoxyribonucleoside triphosphates and a modified Taq polymerase. The dye-labeled primer is extended one base by the dye-terminator specific for the allele present on the template. At the end of the genotyping reaction, the fluorescence intensities of the two dyes in the reaction mixture are analyzed directly without separation or purification. All of these steps can be performed in the same tube and the fluorescence changes can be monitored in real time. Alternatively, the extended primer may be analyzed by MALDI-TOF Mass Spectrometry. The base at the polymorphic site is identified by the mass added onto the microsequencing primer (see Haff L. A. and Smirnov I. P., Genome Research, 7:378-388, 1997, the disclosure of which is incorporated herein by reference in its entirety).

Microsequencing may be achieved by the established microsequencing method or by developments or derivatives thereof. Alternative methods include several solid-phase microsequencing techniques. The basic microsequencing protocol is the same as described previously, except that the method is conducted as a heterogenous phase assay, in which the primer or the target molecule is immobilized or captured onto a solid support. To simplify the primer separation and the terminal nucleotide addition analysis, oligonucleotides are attached to solid supports or are modified in such ways that permit affinity separation as well as polymerase extension. The 5′ ends and internal nucleotides of synthetic oligonucleotides can be modified in a number of different ways to permit different affinity separation approaches, e.g., biotinylation. If a single affinity group is used on the oligonucleotides, the oligonucleotides can be separated from the incorporated terminator regent. This eliminates the need of physical or size separation. More than one oligonucleotide can be separated from the terminator reagent and analyzed simultaneously if more than one affinity group is used. This permits the analysis of several nucleic acid species or more nucleic acid sequence information per extension reaction. The affinity group need not be on the priming oligonucleotide but could alternatively be present on the template. For example, immobilization can be carried out via an interaction between biotinylated DNA and streptavidin-coated microtitration wells or avidin-coated polystyrene particles. In the same manner oligonucleotides or templates may be attached to a solid support in a high-density format. In such solid phase microsequencing reactions, incorporated ddNTPs can be radiolabeled (Syvänen, Clinica Chimica Acta 226:225-236, 1994, the disclosure of which is incorporated herein by reference in its entirety), or linked to fluorescein (Livak and Hainer, Human Mutation 3:379-385,1994, the disclosure of which is incorporated herein by reference in its entirety). The detection of radiolabeled ddNTPs can be achieved through scintillation-based techniques. The detection of fluorescein-linked ddNTT's can be based on the binding of antifluorescein antibody conjugated with alkaline phosphatase, followed by incubation with a chromogenic substrate (such asp-nitrophenyl phosphate). Other possible reporter-detection pairs include: ddNTP linked to dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., Clin. Chem. 39/11 2282-2287, 1993, the disclosure of which is incorporated herein by reference in its entirety), or biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate (WO 92/15712, the disclosure of which is incorporated herein by reference in its entirety). As yet another alternative solid-phase microsequencing procedure, Nyren et al. (Analytical Biochemistry 208:171-175, 1993, the disclosure of which is incorporated herein by reference in its entirety), described a method relying on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA).

Pastinen et al. (Genome Research 7:606-614, 1997, the disclosure of which is incorporated herein by reference in its entirety), describe a method for multiplex detection of single nucleotide polymorphism in which the solid phase minisequencing principle is applied to an oligonucleotide array format. High-density arrays of DNA probes attached to a solid support (DNA chips) are further described in III.C.5.

In one aspect, the present invention provides polynucleotides and methods to genotype one or more biallelic markers of the present invention by performing a microsequencing assay. In the preferred embodiment, it will be appreciated that any primer having a 3′ end immediately adjacent to a polymorphic nucleotide may be used as a microsequencing primer. Similarly, it will be appreciated that microsequencing analysis may be performed for any biallelic marker or any combination of biallelic markers of the present invention. One aspect of the present invention is a solid support which includes one or more microsequencing primers comprising nucleotides complementary to the nucleotide sequences of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908 or the complements thereof, or fragments comprising at least 8, at least 12, at least 15, or at least 20 consecutive nucleotides thereof and having a 3′ terminus immediately upstream of the corresponding biallelic marker, for determining the identity of a nucleotide at biallelic marker site.

3) Mismatch Detection Assays Based on Polymerases and Ligases

In one aspect the present invention provides polynucleotides and methods to determine the allele of one or more biallelic markers of the present invention in a biological sample, by mismatch detection assays based on polymerases and/or ligases. These assays are based on the specificity of polymerases and ligases. Polymerization reactions places particularly stringent requirements on correct base pairing of the 3′ end of the amplification primer and the joining of two oligonucleotides hybridized to a target DNA sequence is quite sensitive to mismatches close to the ligation site, especially at the 3′ end. The terms “enzyme based mismatch detection assay” are used herein to refer to any method of determining the allele of a biallelic marker based on the specificity of ligases and polymerases. Preferred methods are described below. Methods, primers and various parameters to amplify DNA fragments comprising biallelic markers of the present invention are further described above in III.B.

Allele Specific Amplification

Discrimination between the two alleles of a biallelic marker can also be achieved by allele specific amplification, a selective strategy, whereby one of the alleles is amplified without amplification of the other allele. This is accomplished by placing a polymorphic base at the 3′ end of one of the amplification primers. Because the extension forms from the 3′ end of the primer, a mismatch at or near this position has an inhibitory effect on amplification. Therefore, under appropriate amplification conditions, these primers only direct amplification on their complementary allele. Designing the appropriate allele-specific primer and the corresponding assay conditions are well with the ordinary skill in the art.

Ligation/Amplification Based Methods

The “Oligonucleotide Ligation Assay” (OLA) uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules. One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected. OLA is capable of detecting biallelic markers and may be advantageously combined with PCR as described by Nickerson D. A. et al. (Proc. Natl. Acad. Sci. U.S.A. 87:8923-8927, 1990, the disclosure of which is incorporated herein by reference in its entirety). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.

Other methods which are particularly suited for the detection of biallelic markers include LCR (ligase chain reaction), Gap LCR (GLCR) which are described above in III.B. As mentioned above LCR uses two pairs of probes to exponentially amplify a specific target. The sequences of each pair of oligonucleotides, is selected to permit the pair to hybridize to abutting sequences of the same strand of the target. Such hybridization forms a substrate for a template-dependant ligase. In accordance with the present invention, LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a biallelic marker site. In one embodiment, either oligonucleotide will be designed to include the biallelic marker site. In such an embodiment, the reaction conditions are selected such that the oligonucleotides can be ligated together only if the target molecule either contains or lacks the specific nucleotide(s) that is complementary to the biallelic marker on the oligonucleotide. In an alternative embodiment, the oligonucleotides will not include the biallelic marker, such that when they hybridize to the target molecule, a “gap” is created as described in WO 90/01069, the disclosure of which is incorporated herein by reference in its entirety. This gap is then “filled” with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides. Thus at the end of each cycle, each single strand has a complement capable of serving as a target during the next cycle and exponential allele-specific amplification of the desired sequence is obtained.

Ligase/Polymerase-mediated Genetic Bit Analysis™ is another method for determining the identity of a nucleotide at a preselected site in a nucleic acid molecule (WO 95/21271, the disclosure of which is incorporated herein by reference in its entirety). This method involves the incorporation of a nucleoside triphosphate that is complementary to the nucleotide present at the preselected site onto the terminus of a primer molecule, and their subsequent ligation to a second oligonucleotide. The reaction is monitored by detecting a specific label attached to the reaction's solid phase or by detection in solution.

4) Hybridization Assay Methods

A preferred method of determining the identity of the nucleotide present at a biallelic marker site involves nucleic acid hybridization. The hybridization probes, which can be conveniently used in such reactions, preferably include the probes defined herein. Any hybridization assay may be used including Southern hybridization, Northern hybridization, dot blot hybridization and solid-phase hybridization (see Sambrook et al., Molecular Cloning—A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., 1989, the disclosure of which is incorporated herein by reference in its entirety).

Hybridization refers to the formation of a duplex structure by two single stranded nucleic acids due to complementary base pairing. Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain minor regions of mismatch. Specific probes can be designed that hybridize to one form of a biallelic marker and not to the other and therefore are able to discriminate between different allelic forms. Allele-specific probes are often used in pairs, one member of a pair showing perfect match to a target sequence containing the original allele and the other showing a perfect match to the target sequence containing the alternative allele. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Stringent, sequence specific hybridization conditions, under which a probe will hybridize only to the exactly complementary target sequence are well known in the art (Sambrook et al., Molecular Cloning—A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., 1989, the disclosure of which is incorporated herein by reference in its entirety). Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. By way of example and not limitation, procedures using conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 h at 65° C., the preferred hybridization temperature, in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpm of ³²P-labeled probe. Alternatively, the hybridization step can be performed at 65° C. in the presence of SSC buffer, 1×SSC corresponding to 0.15M NaCl and 0.05 M Na citrate. Subsequently, filter washes can be done at 37° C. for 1 h in a solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA, followed by a wash in 0.1×SSC at 50° C. for 45 min. Alternatively, filter washes can be performed in a solution containing 2×SSC and 0.1% SDS, or 0.5×SSC and 0.1% SDS, or 0.1×SSC and 0.1% SDS at 68° C. for 15 minute intervals. Following the wash steps, the hybridized probes are detectable by autoradiography. By way of example and not limitation, procedures using conditions of intermediate stringency are as follows: Filters containing DNA are prehybridized, and then hybridized at a temperature of 60° C. in the presence of a 5×SSC buffer and labeled probe. Subsequently, filters washes are performed in a solution containing 2×SSC at 50° C. and the hybridized probes are detectable by autoradiography. Other conditions of high and intermediate stringency which may be used are well known in the art and as cited in Sambrook et al. (Molecular Cloning—A Laboratory Manual, Second Edition, Cold Spring Harbor Press, N.Y., 1989) and Ausubel et al. (Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y., 1989, the disclosure of which is incorporated herein by reference in its entirety).

Although such hybridizations can be performed in solution, it is preferred to employ a solid-phase hybridization assay. The target DNA comprising a biallelic marker of the present invention may be amplified prior to the hybridization reaction. The presence of a specific allele in the sample is determined by detecting the presence or the absence of stable hybrid duplexes formed between the probe and the target DNA. The detection of hybrid duplexes can be carried out by a number of methods. Various detection assay formats are well known which utilize detectable labels bound to either the target or the probe to enable detection of the hybrid duplexes. Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected. Those skilled in the art will recognize that wash steps may be employed to wash away excess target DNA or probe. Standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the primers and probes.

Two recently developed assays allow hybridization-based allele discrimination with no need for separations or washes (see Landegren U. et al., Genome Research, 8:769-776, 1998, the disclosure of which is incorporated herein by reference in its entirety). The TaqMan assay takes advantage of the 5′ nuclease activity of Taq DNA polymerase to digest a DNA probe annealed specifically to the accumulating amplification product. TaqMan probes are labeled with a donor-acceptor dye pair that interacts via fluorescence energy transfer. Cleavage of the TaqMan probe by the advancing polymerase during amplification dissociates the donor dye from the quenching acceptor dye, greatly increasing the donor fluorescence. All reagents necessary to detect two allelic variants can be assembled at the beginning of the reaction and the results are monitored in real time (see Livak et al., Nature Genetics, 9:341-342, 1995, the disclosure of which is incorporated herein by reference in its entirety). In an alternative homogeneous hybridization-based procedure, molecular beacons are used for allele discriminations. Molecular beacons are hairpin-shaped oligonucleotide probes that report the presence of specific nucleic acids in homogeneous solutions. When they bind to their targets, they undergo a conformational reorganization that restores the fluorescence of an internally quenched fluorophore (Tyagi et al., Nature Biotechnology, 16:49-53, 1998, the disclosure of which is incorporated herein by reference in its entirety).

The polynucleotides provided herein can be used in hybridization assays for the detection of biallelic marker alleles in biological samples. These probes are characterized in that they preferably comprise between 8 and 50 nucleotides, and in that they are sufficiently complementary to a sequence comprising a biallelic marker of the present invention to hybridize thereto and preferably sufficiently specific to be able to discriminate the targeted sequence for only one nucleotide variation. The GC content in the probes of the invention usually ranges between 10 and 75%, preferably between 35 and 60%, and more preferably between 40 and 55%. The length of these probes can range from 10, 15, 20, or 30 to at least 100 nucleotides, preferably from 10 to 50, more preferably from 18 to 35 nucleotides. A particularly preferred probe is 25 nucleotides in length. Preferably the biallelic marker is within 4 nucleotides of the center of the polynucleotide probe. In particularly preferred probes the biallelic marker is at the center of said polynucleotide. Shorter probes may lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer probes are expensive to produce and can sometimes self-hybridize to form hairpin structures. Methods for the synthesis of oligonucleotide probes have been described above and can be applied to the probes of the present invention.

Preferably, the probes of the-present invention are labeled or immobilized on a solid support. Labels and solid supports are further described in I. Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example peptide nucleic acids which are disclosed in International Patent Application WO 92/20702, the disclosure of which is hereby incorporated herein by reference in its entirety, and morpholino analogs which are described in U.S. Pat. Nos. 5,185,444; 5,034,506 and 5,142,047, the disclosures of which are hereby incorporated herein by reference in their entireties. The probe may have to be rendered “non-extendable” in that additional dNTPs cannot be added to the probe. In and of themselves analogs usually are non-extendable and nucleic acid probes can be rendered non-extendable by modifying the 3′ end of the probe such that the hydroxyl group is no longer capable of participating in elongation. For example, the 3′ end of the probe can be finctionalized with the capture or detection label to thereby consume or otherwise block the hydroxyl group. Alternatively, the 3′ hydroxyl group simply can be cleaved, replaced or modified, U.S. patent application Ser. No. 07/049,061 filed Apr. 19, 1993 describes modifications, which can be used to render a probe non-extendable.

The probes of the present invention are useful for a number of purposes. They can be used in Southern hybridization to genomic DNA or Northern hybridization to mRNA. The probes can also be used to detect PCR amplification products. By assaying the hybridization to an allele specific probe, one can detect the presence or absence of a biallelic marker allele in a given sample.

High-throughput parallel hybridizations in array format are specifically encompassed within “hybridization assays” and are described below.

Hybridization to Addressable Arrays of Oligonucleotides

Hybridization assays based on oligonucleotide arrays rely on the differences in hybridization stability of short oligonucleotides to perfectly matched and mismatched target sequence variants. Efficient access to polymorphism information is obtained through a basic structure comprising high-density arrays of oligonucleotide probes attached to a solid support (the chip) at selected positions. Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged in a grid-like pattern and miniaturized to the size of a dime.

The chip technology has already been applied with success in numerous cases. For example, the screening of mutations has been undertaken in the BRCA1 gene, in S. cerevisiae mutant strains, and in the protease gene of HIV-1 virus (Hacia et al., Nature Genetics, 14(4):441-447, 1996; Shoemaker et al., Nature Genetics, 14(4):450-456, 1996; Kozal et al., Nature Medicine, 2:753-759, 1996, the disclosures of which are incorporated herein by reference in their entireties). Chips of various formats for use in detecting biallelic polymorphisms can be produced on a customized basis by Affymetrix (GeneChip™), Hyseq (HyChip and HyGnostics), and Protogene Laboratories.

In general, these methods employ arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual which, target sequences include a polymorphic marker. EP785280, the disclosure of which is incorporated herein by reference in its entirety, describes a tiling strategy for the detection of single nucleotide polymorphisms. Briefly, arrays may generally be “tiled” for a large number of specific polymorphisms. By “tiling” is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of monomers, i.e. nucleotides. Tiling strategies are further described in PCT application No. WO 95/11995, the disclosure of which is incorporated herein by reference in its entirety. In a particular aspect, arrays are tiled for a number of specific, identified biallelic marker sequences. In particular the array is tiled to include a number of detection blocks, each detection block being specific for a specific biallelic marker or a set of biallelic markers. For example, a detection block may be tiled to include a number of probes, which span the sequence segment that includes a specific polymorphism. To ensure probes that are complementary to each allele, the probes are synthesized in pairs differing at the biallelic marker. In addition to the probes differing at the polymorphic base, monosubstituted probes are also generally tiled within the detection block. These monosubstituted probes have bases at and up to a certain number of bases in either direction from the polymorphism, substituted with the remaining nucleotides (selected from A, T, G, C and U). Typically the probes in a tiled detection block will include substitutions of the sequence positions up to and including those that are 5 bases away from the biallelic marker. The monosubstituted probes provide internal controls for the tiled array, to distinguish actual hybridization from artefactual cross-hybridization. Upon completion of hybridization with the target sequence and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes. The hybridization data from the scanned array is then analyzed to identify which allele or alleles of the biallelic marker are present in the sample. Hybridization and scanning may be carried out as described in PCT application No. WO 92/10092 and WO 95/11995 and U.S. Pat. No. 5,424,186, the disclosures of which are incorporated herein by reference in their entireties.

Thus, in some embodiments, the chips may comprise an array of nucleic acid sequences of fragments of about 15 nucleotides in length. In further embodiments, the chip may comprise an array including at least one of the sequences selected from the group consisting of SEQ ID No. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908 and the sequences complementary thereto, or a fragment thereof at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably least 30, 35, 43, 44, 45, 46 or 47 consecutive nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID. In some embodiments, the chip may comprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these polynucleotides of the invention. Solid supports and polynucleotides of the present invention attached to solid supports are further described in I.

5) Integrated Systems

Another technique, which may be used to analyze polymorphisms, includes multicomponent integrated systems, which miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device. An example of such technique is disclosed in U.S. Pat. No. 5,589,136, the disclosure of which is hereby incorporated herein by reference in its entirety, which describes the integration of PCR amplification and capillary electrophoresis in chips.

Integrated systems can be envisaged mainly when microfluidic systems are used. These systems comprise a pattern of microchannels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip. The movements of the samples are controlled by electric, electroosmotic or hydrostatic forces applied across different areas of the microchip. For genotyping biallelic markers, the microfluidic system may integrate nucleic acid amplification, microsequencing, capillary electrophoresis and a detection method such as laser-induced fluorescence detection.

IV. Methods Of Genetic Analysis Using The Biallelic Markers Of The Present Invention

Different methods are available for the genetic analysis of complex traits (see Lander and Schork, Science, 265, 2037-2048, 1994, the disclosure of which is hereby incorporated herein by reference in its entirety). The search for disease-susceptibility genes is conducted using two main methods: the linkage approach in which evidence is sought for cosegregation between a locus and a putative trait locus using family studies, and the association approach in which evidence is sought for a statistically significant association between an allele and a trait or a trait causing allele (Khoury J. et al., Fundamentals of Genetic Epidemiology, Oxford University Press, New York, 1993, the disclosure of which is incorporated herein by reference in its entirety). In general, the biallelic markers of the present invention find use in any method known in the art to demonstrate a statistically significant correlation between a genotype and a phenotype. The biallelic markers may be used in parametric and non-parametric linkage analysis methods. Preferably, the biallelic markers of the present invention are used to identify genes associated with detectable traits using association studies, an approach which does not require the use of affected families and which permits the identification of genes associated with complex and sporadic traits.

The genetic analysis using the biallelic markers of the present invention may be conducted on any scale. The whole set of biallelic markers of the present invention or any subset of biallelic markers of the present invention may be used. In some embodiments a subset of biallelic markers corresponding to one or several candidate genes may be used. In other embodiments a subset of biallelic markers corresponding to candidate genes from a particular disease pathway may be used. Alternatively, a subset of biallelic markers of the present invention localised on a specific chromosome segment may be used. Further, any set of genetic markers including a biallelic marker of the present invention may be used. A set of biallelic polymorphisms that, could be used as genetic markers in combination with the biallelic markers of the present invention, has been described in WO 98/20165, the disclosure of which is incorporated herein by reference in its entirety. As mentioned above, it should be noted that the biallelic markers of the present invention may be included in any complete or partial genetic map of the human genome. These different uses are specifically contemplated in the present invention and claims.

IV.A. Linkage Analysis

Linkage analysis is based upon establishing a correlation between the transmission of genetic markers and that of a specific trait throughout generations within a family. Thus, the aim of linkage analysis is to detect marker loci that show cosegregation with a trait of interest in pedigrees.

Parametric Methods

When data are available from successive generations there is the opportunity to study the degree of linkage between pairs of loci. Estimates of the recombination fraction enable loci to be ordered and placed onto a genetic map. With loci that are genetic markers, a genetic map can be established, and then the strength of linkage between markers and traits can be calculated and used to indicate the relative positions of markers and genes affecting those traits (Weir, B. S., Genetic data Analysis II: Methods for Discrete population genetic Data, Sinauer Assoc., Inc., Sunderland, Mass., USA, 1996, the disclosure of which is incorporated herein by reference in its entirety). The classical method for linkage analysis is the logarithm of odds (lod) score method (see Morton N. E., Am. J. Hum. Genet., 7:277-318, 1955; Ott J., Analysis of Human Genetic Linkage, John Hopkins University Press, Baltimore, 1991, the disclosures of which are incorporated herein by reference in their entireties). Calculation of lod scores requires specification of the mode of inheritance for the disease (parametric method). Generally, the length of the candidate region identified using linkage analysis is between 2 and 20 Mb. Once a candidate region is identified as described above, analysis of recombinant individuals using additional markers allows further delineation of the candidate region. Linkage analysis studies have generally relied on the use of a maximum of 5,000 microsatellite markers, thus limiting the maximum theoretical attainable resolution of linkage analysis to about 600 kb on average.

Linkage analysis has been successfully applied to map simple genetic traits that show clear Mendelian inheritance patterns and which have a high penetrance (i.e., the ratio between the number of trait positive carriers of allele a and the total number of a carriers in the population). However, parametric linkage analysis suffers from a variety of drawbacks. First, it is limited by its reliance on the choice of a genetic model suitable for each studied trait. Furthermore, as already mentioned, the resolution attainable using linkage analysis is limited, and complementary studies are required to refine the analysis of the typical 2 Mb to 20 Mb regions initially identified through linkage analysis. In addition, parametric linkage analysis approaches have proven difficult when applied to complex genetic traits, such as those due to the combined action of multiple genes and/or environmental factors. It is very difficult to model these factors adequately in a lod score analysis. In such cases, too large an effort and cost are needed to recruit the adequate number of affected families required for applying linkage analysis to these situations, as recently discussed by Risch, N. and Merikangas, K. (Science, 273:1516-1517, 1996, the disclosure of which is incorporated herein by reference in its entirety).

Non-parametric Methods

The advantage of the so-called non-parametric methods for linkage analysis is that they do not require specification of the mode of inheritance for the disease, they tend to be more useful for the analysis of complex traits. In non-parametric methods, one tries to prove that the inheritance pattern of a chromosomal region is not consistent with random Mendelian segregation by showing that affected relatives inherit identical copies of the region more often than expected by chance. Affected relatives should show excess “allele sharing” even in the presence of incomplete penetrance and polygenic inheritance. In non-parametric linkage analysis the degree of agreement at a marker locus in two individuals can be measured either by the number of alleles identical by state (IBS) or by the number of alleles identical by descent (IBD). Affected sib pair analysis is a well-known special case and is the simplest form of these methods.

The biallelic markers of the present invention may be used in both parametric and non-parametric linkage analysis. Preferably biallelic markers may be used in non-parametric methods which allow the mapping of genes involved in complex traits. The biallelic markers of the present invention may be used in both IBD- and IBS- methods to map genes affecting a complex trait. In such studies, taking advantage of the high density of biallelic markers, several adjacent biallelic marker loci may be pooled to achieve the efficiency attained by multi-allelic markers (Zhao et al., Am. J. Hum. Genet., 63:225-240, 1998, the disclosure of which is incorporated herein by reference in its entirety).

However, both parametric and non-parametric linkage analysis methods analyse affected relatives, they tend to be of limited value in the genetic analysis of drug responses or in the analysis of side effects to treatments. This type of analysis is impractical in such cases due to the lack of availability of familial cases. In fact, the likelihood of having more than one individual in a family being exposed to the same drug at the same time is extremely low.

IV.B. Population Association Studies

The present invention comprises methods for identifying one or several genes among a set of candidate genes that are associated with a detectable trait using the biallelic markers of the present invention. In one embodiment the present invention comprises methods to detect an association between a biallelic marker allele or a biallelic marker haplotype and a trait. Further, the invention comprises methods to identify a trait causing allele in linkage disequilibrium with any biallelic marker allele of the present invention.

As described above, alternative approaches can be employed to perform association studies: genome-wide association studies, candidate region association studies and candidate gene association studies. In a preferred embodiment, the biallelic markers of the present invention are used to perform candidate gene association studies. Further, the biallelic markers of the present invention may be incorporated in any map of genetic markers of the human genome in order to perform genome-wide association studies. Methods to generate a high-density map of biallelic markers has been described in US Provisional Patent application serial No. 60/082,614. The biallelic markers of the present invention may further be incorporated in any map of a specific candidate region of the genome (a specific chromosome or a specific chromosomal segment for example).

As mentioned above, association studies may be conducted within the general population and are not limited to studies performed on related individuals in affected families. Association studies are extremely valuable as they permit the analysis of sporadic or multifactor traits. Moreover, association studies represent a powerful method for fine-scale mapping enabling much finer mapping of trait causing alleles than linkage studies. Studies based on pedigrees often only narrow the location of the trait causing allele. Association studies using the biallelic markers of the present invention can therefore be used to refine the location of a trait causing allele in a candidate region identified by Linkage Analysis methods. Moreover, once a chromosome segment of interest has been identified, the presence of a candidate gene such as a candidate gene of the present invention, in the region of interest can provide a shortcut to the identification of the trait causing allele. Biallelic markers of the present invention can be used to demonstrate that a candidate gene is associated with a trait. Such uses are specifically contemplated in the present invention and claims.

1) Determining the Frequency of a Biallelic Marker Allele or of a Biallelic Marker Haplotype in a Population

Association studies explore the relationships among frequencies for sets of alleles between loci.

Determining the Frequency of an Allele in a Population

Allelic frequencies of the biallelic markers in a population can be determined using one of the methods described above under the heading “Methods for genotyping an individual for biallelic markers”, or any genotyping procedure suitable for this intended purpose. Genotyping pooled samples or individual samples can determine the frequency of a biallelic marker allele in a population. One way to reduce the number of genotypings required is to use pooled samples. A major obstacle in using pooled samples is in terms of accuracy and reproducibility for determining accurate DNA concentrations in setting up the pools. Genotyping individual samples provides higher sensitivity, reproducibility and accuracy and; is the preferred method used in the present invention. Preferably, each individual is genotyped separately and simple gene counting is applied to determine the frequency of an allele of a biallelic marker or of a genotype in a given population.

Determining the Frequency of a Haplotype in a Population

The gametic phase of haplotypes is unknown when diploid individuals are heterozygous at more than one locus. Using genealogical information in families gametic phase can sometimes be inferred (Perlin et al., Am. J. Hum. Genet., 55:777-787, 1994, the disclosure of which is incorporated herein by reference in its entirety). When no genealogical information is available different strategies may be used. One possibility is that the multiple-site heterozygous diploids can be eliminated from the analysis, keeping only the homozygotes and the single-site heterozygote individuals, but this approach might lead to a possible bias in the sample composition and the underestimation of low-frequency haplotypes. Another possibility is that single chromosomes can be studied independently, for example, by asymmetric PCR amplification (see Newton et al., Nucleic Acids Res., 17:2503-2516, 1989; Wu et al., Proc. Natl. Acad. Sci. USA, 86:2757, 1989, the disclosures of which are incorporated herein by reference in their entireties) or by isolation of single chromosome by limit dilution followed by PCR amplification (see Ruano et al., Proc. Natl. Acad. Sci. USA, 87:6296-6300, 1990, the disclosure of which is incorporated herein by reference in its entirety). Further, a sample may be haplotyped for sufficiently close biallelic markers by double PCR amplification of specific alleles (Sarkar, G. and Sommer S. S., Biotechniques, 1991, the disclosure of which is incorporated herein by reference in its entirety). These approaches are not entirely satisfying either because of their technical complexity, the additional cost they entail, their lack of generalisation at a large scale, or the possible biases they introduce. To overcome these difficulties, an algorithm to infer the phase of PCR-amplified DNA genotypes introduced by Clark A. G. (Mol. Biol. Evol, 7:111-122, 1990, the disclosure of which is incorporated herein by reference in its entirety) may be used. Briefly, the principle is to start filling a preliminary list of haplotypes present in the sample by examining unambiguous individuals, that is, the complete homozygotes and the single-site heterozygotes. Then other individuals in the same sample are screened for the possible occurrence of previously recognised haplotypes. For each positive identification, the complementary haplotype is added to the list of recognised haplotypes, until the phase information for all individuals is either resolved or identified as unresolved. This method assigns a single haplotype to each multiheterozygous individual, whereas several haplotypes are possible when there are more than one heterozygous site. Alternatively, one can use methods estimating haplotype frequencies in a population without assigning haplotypes to each individual. Preferably, a method based on an expectation-maximization (EM) algorithm (Dempster et al., J. R. Stat. Soc., 39B: 1-38, 1977, the disclosure of which is incorporated herein by reference in its entirety) leading to maximum-likelihood estimates of haplotype frequencies under the assumption of Hardy-Weinberg proportions (random mating) is used (see Excoffier L. and Slatkin M., Mol. Biol. Evol., 12(5): 921-927, 1995, the disclosure of which is incorporated herein by reference in its entirety). The EM algorithm is a generalised iterative maximum-likelihood approach to estimation that is useful when data are ambiguous and/or incomplete. The EM algorithm is used to resolve heterozygotes into haplotypes. Haplotype estimations are further described below under the heading “Statistical methods”. Any other method known in the art to determine or to estimate the frequency of a haplotype in a population may also be used.

2) Linkage Disequilibrium Analysis

Linkage disequilibrium is the non-random association of alleles at two or more loci and represents a powerful tool for mapping genes involved in disease traits (see Ajioka R. S. et al., Am. J. Hum. Genet., 60:1439-1447, 1997, the disclosure of which is incorporated herein by reference in its entirety). Biallelic markers, because they are densely spaced in the human genome and can be genotyped in more numerous numbers than other types of genetic markers (such as RFLP or VNTR markers), are particularly useful in genetic analysis based on linkage disequilibrium. The biallelic markers of the present invention may be used in any linkage disequilibrium analysis method known in the art.

Briefly, when a disease mutation is first introduced into a population (by a new mutation or the immigration of a mutation carrier), it necessarily resides on a single chromosome and thus on a single “background” or “ancestral” haplotype of linked markers. Consequently, there is complete disequilibrium between these markers and the disease mutation: one finds the disease mutation only in the presence of a specific set of marker alleles. Through subsequent generations recombinations occur between the disease mutation and these marker polymorphisms, and the disequilibrium gradually dissipates. The pace of this dissipation is a function of the recombination frequency, so the markers closest to the disease gene will manifest higher levels of disequilibrium than those that are further away. When not broken up by recombination, “ancestral” haplotypes and linkage disequilibrium between marker alleles at different loci can be tracked not only through pedigrees but also through populations. Linkage disequilibrium is usually seen as an association between one specific allele at one locus and another specific allele at a second locus.

The pattern or curve of disequilibrium between disease and marker loci is expected to exhibit a maximum that occurs at the disease locus. Consequently, the amount of linkage disequilibrium between a disease allele and closely linked genetic markers may yield valuable information regarding the location of the disease gene. For fine-scale mapping of a disease locus, it is useful to have some knowledge of the patterns of linkage disequilibrium that exist between markers in the studied region. As mentioned above the mapping resolution achieved through the analysis of linkage disequilibrium is much higher than that of linkage studies. The high density of biallelic markers combined with linkage disequilibrium analysis provides powerful tools for fine-scale mapping. Different methods to calculate linkage disequilibrium are described below under the heading “Statistical Methods”.

3) Population-based Case-control Studies of Trait-marker Associations

As mentioned above, the occurrence of pairs of specific alleles at different loci on the same chromosome is not random and the deviation from random is called linkage disequilibrium. Association studies focus on population frequencies and rely on the phenomenon of linkage disequilibrium. If a specific allele in a given gene is directly involved in causing a particular trait, its frequency will be statistically increased in an affected (trait positive) population, when compared to the frequency in a trait negative population or in a random control population. As a consequence of the existence of linkage disequilibrium, the frequency of all other alleles present in the haplotype carrying the trait-causing allele will also be increased in trait positive individuals compared to trait negative individuals or random controls. Therefore, association between the trait and any allele (specifically a biallelic marker allele) in linkage disequilibrium with the trait-causing allele will suffice to suggest the presence of a trait-related gene in that particular region. Case-control populations can be genotyped for biallelic markers to identify associations that narrowly locate a trait causing allele. As any marker in linkage disequilibrium with one given marker associated with a trait will be associated with the trait. Linkage disequilibrium allows the relative frequencies in case-control populations of a limited number of genetic polymorphisms (specifically biallelic markers) to be analysed as an alternative to screening all possible functional polymorphisms in order to find trait-causing alleles. Association studies compare the frequency of marker alleles in unrelated case-control populations, and represent powerful tools for the dissection of complex traits.

Case-control Populations (Inclusion Criteria)

Population-based association studies do not concern familial inheritance but compare the prevalence of a particular genetic marker, or a set of markers, in case-control populations. They are case-control studies based on comparison of unrelated case (affected or trait positive) individuals and unrelated control (unaffected or trait negative or random) individuals. Preferably the control group is composed of unaffected or trait negative individuals. Further, the control group is ethnically matched to the case population. Moreover, the control group is preferably matched to the case-population for the main known confusion factor for the trait under study (for example age-matched for an age-dependent trait). Ideally, individuals in the two samples are paired in such a way that they are expected to differ only in their disease status. In the following “trait positive population”, “case population” and “affected population” are used interchangeably.

An important step in the dissection of complex traits using association studies is the choice of case-control populations (see Lander and Schork, Science, 265, 2037-2048, 1994, the disclosure of which is incorporated herein by reference in its entirety). A major step in the choice of case-control populations is the clinical definition of a given trait or phenotype. Any genetic trait may be analysed by the association method proposed here by carefully selecting the individuals to be included in the trait positive and trait negative phenotypic groups. Four criteria are often useful: clinical phenotype, age at onset, family history and severity. The selection procedure for continuous or quantitative traits (such as blood pressure for example) involves selecting individuals at opposite ends of the phenotype distribution of the trait under study, so as to include in these trait positive and trait negative populations individuals with non-overlapping phenotypes. Preferably, case-control populations consist of phenotypically homogeneous populations. Trait positive and trait negative populations consist of phenotypically uniform populations of individuals representing each between 1 and 98%, preferably between 1 and 80%, more preferably between 1 and 50%, and more preferably between 1 and 30%, most preferably between 1 and 20% of the total population under study, and selected among individuals exhibiting non-overlapping phenotypes. The clearer the difference between the two trait phenotypes, the greater the probability of detecting an association with biallelic markers. The selection of those drastically different but relatively uniform phenotypes enables efficient comparisons in association studies and the possible detection of marked differences at the genetic level, provided that the sample sizes of the populations under study are significant enough.

In preferred embodiments, a first group of between 50 and 300 trait positive individuals, preferably about 100 individuals, are recruited according to their phenotypes. A similar number of trait negative individuals are included in such studies.

Association Analysis

The general strategy to perform association studies using biallelic markers derived from a region carrying a candidate gene is to scan two groups of individuals (case-control populations) in order to measure and statistically compare the allele frequencies of the biallelic markers of the present invention in both groups.

If a statistically significant association with a trait is identified for at least one or more of the analysed biallelic markers, one can assume that: either the associated allele is directly responsible for causing the trait (the associated allele is the trait causing allele), or more likely the associated allele is in linkage disequilibrium with the trait causing allele. The specific characteristics of the associated allele with respect to the candidate gene function usually gives further insight into the relationship between the associated allele and the trait (causal or in linkage disequilibrium). If the evidence indicates that the associated allele within the candidate gene is most probably not the trait causing allele but is in linkage disequilibrium with the real trait causing allele, then the trait causing allele can be found by sequencing the vicinity of the associated marker.

Association studies are usually run in two successive steps. In a first phase, the frequencies of a reduced number of biallelic markers from one or several candidate genes are determined in the trait positive and trait negative populations. In a second phase of the analysis, the identity of the candidate gene and the position of the genetic loci responsible for the given trait is further refined using a higher density of markers from the relevant region. However, if the candidate gene under study is relatively small in length, as it is the case for many of the candidate genes analysed included in the present invention, a single phase may be sufficient to establish significant associations.

Haplotype Analysis

As described above, when a chromosome carrying a disease allele first appears in a population as a result of either mutation or migration, the mutant allele necessarily resides on a chromosome having a set of linked markers: the ancestral haplotype. This haplotype can be tracked through populations and its statistical association with a given trait can be analysed. Complementing single point (allelic) association studies with multi-point association studies also called haplotype studies increases the statistical power of association studies. Thus, a haplotype association study allows one to define the frequency and the type of the ancestral carrier haplotype. A haplotype analysis is important in that it increases the statistical power of an analysis involving individual markers.

In a first stage of a haplotype frequency analysis, the frequency of the possible haplotypes based on various combinations of the identified biallelic markers of the invention is determined. The haplotype frequency is then compared for distinct populations of trait positive and control individuals. The number of trait positive individuals, which should be, subjected to this analysis to obtain statistically significant results usually ranges between 30 and 300, with a preferred number of individuals ranging between 50 and 150. The same considerations apply to the number of unaffected individuals (or random control) used in the study. The results of this first analysis provide haplotype frequencies in case-control populations, for each evaluated haplotype frequency a p-value and an odd ratio are calculated. If a statistically significant association is found the relative risk for an individual carrying the given haplotype of being affected with the trait under study can be approximated.

Interaction Analysis

The biallelic markers of the present invention may also be used to identify patterns of biallelic markers associated with detectable traits resulting from polygenic interactions. The analysis of genetic interaction between alleles at unlinked loci requires individual genotyping using the techniques described herein. The analysis of allelic interaction among a selected set of biallelic markers with appropriate level of statistical significance can be considered as a haplotype analysis. Interaction analysis consists in stratifying the case-control populations with respect to a given haplotype for the first loci and performing a haplotype analysis with the second loci with each subpopulation.

Statistical methods used in association studies are further described below in IV.C.

4) Testing for Linkage in the Presence of Association

The biallelic markers of the present invention may further be used in TDT (transmission/disequilibrium test). TDT tests for both linkage and association and is not affected by population stratification. TDT requires data for affected individuals and their parents or data from unaffected sibs instead of from parents (see Spielmann S. et al., Am. J. Hum. Genet., 52:506-516, 1993; Schaid D. J. et al., Genet. Epidemiol., 13:423-450, 1996, Spielmann S. and Ewens W. J., Am. J. Hum. Genet., 62:450-458, 1998, the disclosures of which are incorporated herein by reference in their entireties). Such combined tests generally reduce the false—positive errors produced by separate analyses.

IV.C. Statistical Methods

In general, any method known in the art to test whether a trait and a genotype show a statistically significant correlation may be used.

1) Methods in Linkage Analysis

Statistical methods and computer programs useful for linkage analysis are well-known to those skilled in the art (see Terwilliger J. D. and Ott J., Handbook of Human Genetic Linkage, John Hopkins University Press, London, 1994; Ott J., Analysis of Human Genetic Linkage, John Hopkins University Press, Baltimore, 1991, the disclosures of which are incorporated herein by reference in their entireties).

2) Methods to Estimate Haplotype Frequencies in a Population

As described above, when genotypes are scored, it is often not possible to distinguish heterozygotes so that haplotype frequencies cannot be easily inferred. When the gametic phase is not known, haplotype frequencies can be estimated from the multilocus genotypic data. Any method known to person skilled in the art can be used to estimate haplotype frequencies (see Lange K., Mathematical and Statistical Methods for Genetic Analysis, Springer, N.Y. 1997; Weir, B. S., Genetic data Analysis II: Methods for Discrete population genetic Data, Sinauer Assoc., Inc., Sunderland, Mass., USA, 1996, the disclosures of which are incorporated herein by reference in their entireties) Preferably, maximum-likelihood haplotype frequencies are computed using an Expectation-Maximization (EM) algorithm (see Dempster et al., J. R. Stat. Soc., 39B:1-38, 1977; Excoffier L. and Slatkin M., Mol. Biol. Evol., 12(5): 921-927, 1995, the disclosures of which are incorporated herein by reference in their entireties). This procedure is an iterative process aiming at obtaining maximum-likelihood estimates of haplotype frequencies from multi-locus genotype data when the gametic phase is unknown. Haplotype estimations are usually performed by applying the EM algorithm using for example the EM-HAPLO program (Hawley M. E. et al., Am. J. Phys. Anthropol., 18:104, 1994, the disclosure of which is incorporated herein by reference in its entirety) or the Arlequin program (Schneider et al., Arlequin: a software for population genetics data analysis, University of Geneva, 1997, the disclosure of which is incorporated herein by reference in its entirety). The EM algorithm is a generalised iterative maximum likelihood approach to estimation and is briefly described below.

In the following part of this text, phenotypes will refer to multi-locus genotypes with unknown phase. Genotypes will refer to known-phase multi-locus genotypes.

Suppose a sample of N unrelated individuals typed for K markers. The data observed are the unknown-phase K-locus phenotypes that can categorised in F different phenotypes. Suppose that we have H underlying possible haplotypes (in case of K biallelic markers, H=2^(K)). For phenotype j, suppose that c_(j) genotypes are possible. We thus have the following equation $\begin{matrix} {{Pj} = {{\sum\limits_{i = 1}^{c_{j}}{{pr}\quad \left( {genotype}_{i} \right)}} = {\sum\limits_{i = 1}^{c_{j}}{{pr}\quad \left( {h_{k},h_{l}} \right)}}}} & {{Equation}\quad 1} \end{matrix}$

where P_(j) is the probability of the phenotypes, h_(k) and h_(l) are the two haplotypes constituent the genotype i. Under the Hardy-Weinberg equilibrium, pr(h_(k)h_(l)) becomes:

pr(h _(k) ,h _(l))=pr(h _(k))² if h _(k) =h _(l) ,pr(h _(k) ,h _(l))=2pr(h _(k)).pr(h _(l)) if h _(k) ≠h _(l).  Equation 2

The successive steps of the E-M algorithm can be described as follows: Starting with initial values of the of haplotypes frequencies, noted p₁ ⁽⁰⁾, p₂ ⁽⁰⁾, . . . P_(H) ⁽⁰⁾, these initial values serve to estimate the genotype frequencies (Expectation step) and then estimate another set of haplotype frequencies (Maximisation step), noted p₁ ⁽¹⁾, p₂ ⁽¹⁾, . . . p_(H) ⁽¹⁾, these two steps are iterated until changes in the sets of haplotypes frequency are very small.

A stop criterion can be that the maximum difference between haplotype frequencies between two iterations is less than 10⁻⁷. These values can be adjusted according to the desired precision of estimations.

In details, at a given iteration s, the Expectation step consists in calculating the genotypes frequencies by the following equation: $\begin{matrix} {{{pr}\quad \left( {genotype}_{i} \right)^{(s)}} = {{{pr}\quad {\left( {phenotype}_{j} \right) \cdot {pr}}\quad \left( {genotype}_{i} \middle| \quad {phenotype}_{j} \right)^{(s)}} = {\frac{n_{j}}{N} \cdot \frac{{pr}\quad \left( {h_{k},h_{l}} \right)^{(s)}}{P_{j}^{(s)}}}}} & {{Equation}\quad 3} \end{matrix}$

where genotype i occurs in phenotype j, and where h_(k) and h_(l) constitute genotype i. Each probability is derived according to eq.1, and eq.2 described above.

Then the Maximisation step simply estimates another set of haplotype frequencies given the genotypes frequencies. This approach is also known as gene-counting method (Smith, Ann. Hum. Genet., 21:254-276, 1957, the disclosure of which is incorporated herein by reference in its entirety). $\begin{matrix} {p_{t}^{({s + 1})} = {\frac{1}{2}{\sum\limits_{j = 1}^{F}{\sum\limits_{i = 1}^{c_{j}}{{\delta_{it} \cdot {pr}}\quad \left( {genotype}_{i} \right)^{(s)}}}}}} & {{Equation}\quad 4} \end{matrix}$

Where δ_(it) is an indicator variable which count the number of time haplotype t in genotype i. It takes the values of 0, 1 or 2.

To ensure that the estimation finally obtained is the maximum-likelihood estimation several values of departures are required. The estimations obtained are compared and if they are different the estimations leading to the best likelihood are kept.

3) Methods to Calculate Linkage Disequilibrium Between Markers

A number of methods can be used to calculate linkage disequilibrium between any two genetic positions, in practice linkage disequilibrium is measured by applying a statistical association test to haplotype data taken from a population.

Linkage disequilibrium between any pair of biallelic markers comprising at least one of the biallelic markers of the present invention (M_(i), M_(j)) having alleles (a_(i)/b_(i)) at marker M_(i) and alleles (a_(j)/b_(j)) at marker M_(j) can be calculated for every allele combination (a_(i),a_(j); a_(i),b_(j); b_(i),a_(j) and b_(i),b_(j)), according to the Piazza formula:

Δ_(aiaj)=θ4−(θ4+θ3)(θ4+θ2), where:

θ4=−−=frequency of genotypes not having allele a_(i) at M_(i) and not having allele a_(j) at M_(j)

θ3=−+=frequency of genotypes not having allele a_(i) at M_(i) and having allele a_(j) at M_(j)

θ2=−+=frequency of genotypes having allele a_(i) at M_(i) and not having allele a_(j) at M_(j)

Linkage disequilibrium (LD) between pairs of biallelic markers (M_(i), M_(j)) can also be calculated for every allele combination (a_(i),a_(j); a_(i),b_(j); b_(i),a_(j) and b_(i),b_(j)), according to the maximum-likelihood estimate (MLE) for delta (the composite genotypic disequilibrium coefficient), as described by Weir (Weir B. S., Genetic Data Analysis, Sinauer Ass. Eds, 1996, the disclosure of which is incorporated herein by reference in its entirety). The MLE for the composite linkage disequilibrium is:

D _(aiaj)=(2n ₁ +n ₂ +n ₃ +n ₄/2)/N−2(pr(a _(i)).pr(a _(j)))

Where n₁=Σ phenotype (a_(i)/a_(i), a_(j)/a_(j)), n₂=Σ phenotype (a_(i)/a_(i), a_(j)/b_(j)), n₃=Σ phenotype (a_(i)/b_(i), a_(j)/a_(j)), n₄=Σ phenotype (a_(i)/b_(i), a_(j)/b_(j)) and N is the number of individuals in the sample. This formula allows linkage disequilibrium between alleles to be estimated when only genotype, and not haplotype, data are available.

Another means of calculating the linkage disequilibrium between markers is as follows. For a couple of biallelic markers, M_(i) (a_(i)/b_(i)) and M_(j) (a_(j)/b_(j)), fitting the Hardy-Weinberg equilibrium, one can estimate the four possible haplotype frequencies in a given population according to the approach described above.

The estimation of gametic disequilibrium between a_(i) and a_(j) is simply:

D _(aiaj) =pr(haplotype(a _(i) ,a _(j)))−pr(a _(i)).pr(a _(j)).

Where pr(a_(i)) is the probability of allele a_(i) and pr(a_(j)) is the probability of allele a_(j) and where pr(haplotype (a_(i),a_(j))) is estimated as in Equation 3 above. For a couple of biallelic marker only one measure of disequilibrium is necessary to describe the association between M_(i) and M_(j).

Then a normalised value of the above is calculated as follows:

D′ _(aiaj) =D _(aiaj)/max(−pr(a _(i)).pr(a _(j)),−pr(b _(i)).pr(b _(j))) with D _(aiaj)<0

D′ _(aiaj) =D _(aiaj)/max(pr(b _(i)).pr(a _(j)),pr(a _(i)).pr(b _(j))) with D _(aiaj)<0

The skilled person will readily appreciate that other LD calculation methods can be used without undue experimentation.

Linkage disequilibrium among a set of biallelic markers having an adequate heterozygosity rate can be determined by genotyping between 50 and 1000 unrelated individuals, preferably between 75 and 200, more preferably around 100.

4) Testing for Association

Methods for determining the statistical significance of a correlation between a phenotype and a genotype, in this case an allele at a biallelic marker or a haplotype made up of such alleles, may be determined by any statistical test known in the art and with any accepted threshold of statistical significance being required. The application of particular methods and thresholds of significance are well with in the skill of the ordinary practitioner of the art.

Testing for association is performed by determining the frequency of a biallelic marker allele in case and control populations and comparing these frequencies with a statistical test to determine if their is a statistically significant difference in frequency which would indicate a correlation between the trait and the biallelic marker allele under study. Similarly, a haplotype analysis is performed by estimating the frequencies of all possible haplotypes for a given set of biallelic markers in case and control populations, and comparing these frequencies with a statistical test to determine if their is a statistically significant correlation between the haplotype and the phenotype (trait) under study. Any statistical tool useful to test for a statistically significant association between a genotype and a phenotype may be used. Preferably the statistical test employed is a chi-square test with one degree of freedom. A p-value is calculated (the p-value is the probability that a statistic as large or larger than the observed one would occur by chance).

Statistical Significance

In preferred embodiments, significance for diagnosis purposes, either as a positive basis for further diagnostic tests or as a preliminary starting point for early preventive therapy, the p value related to a biallelic marker association is preferably about 1×10⁻² or less, more preferably about 1×10⁻⁴ or less, for a single biallelic marker analysis and about 1×10⁻³ or less, still more preferably 1×10⁻⁶ or less and most preferably of about 1×10⁻⁸ or less, for a haplotype analysis involving several markers. These values are believed to be applicable to any association studies involving single or multiple marker combinations.

The skilled person can use the range of values set forth above as a starting point in order to carry out association studies with biallelic markers of the present invention. In doing so, significant associations between the biallelic markers of the present invention and diseases can be revealed.

Phenotypic Permutation

In order to confirm the statistical significance of the first stage haplotype analysis described above, it might be suitable to perform further analyses in which genotyping data from case-control individuals are pooled and randomised with respect to the trait phenotype. Each individual genotyping data is randomly allocated to two groups, which contain the same number of individuals as the case-control populations used to compile the data obtained in the first stage. A second stage haplotype analysis is preferably run on these artificial groups, preferably for the markers included in the haplotype of the first stage analysis showing the highest relative risk coefficient. This experiment is reiterated preferably at least between 100 and 10000 times. The repeated iterations allow the determination of the percentage of obtained haplotypes with a significant p-value level.

Assessment of Statistical Association

To address the problem of false positives similar analysis may be performed with the same case-control populations in random genomic regions. Results in random regions and the candidate region are compared as described in US Provisional Patent Application entitled “Methods, software and apparati for identifying genomic regions harbouring a gene associated with a detectable trait”.

5) Evaluation of Risk Factors

The association between a risk factor (in genetic epidemiology the risk factor is the presence or the absence of a certain allele or haplotype at marker loci) and a disease is measured by the odds ratio (OR) and by the relative risk (RR). If P(R⁺) is the probability of developing the disease for individuals with R and P(R⁻) is the probability for individuals without the risk factor, then the relative risk is simply the ratio of the two probabilities, that is:

RR=P(R ⁺)/P(R ⁻)

In case-control studies, direct measures of the relative risk cannot be obtained because of the sampling design. However, the odds ratio allows a good approximation of the relative risk for low-incidence diseases and can be calculated: ${OR} = {\left\lbrack \frac{F^{+}}{1 - F^{+}} \right\rbrack/\left\lbrack \frac{F^{-}}{\left( {1 - F^{-}} \right)} \right\rbrack}$

F⁺ is the frequency of the exposure to the risk factor in cases and F⁻ is the frequency of the exposure to the risk factor in controls. F⁺ and F⁻ are calculated using the allelic or haplotype frequencies of the study and further depend on the underlying genetic model (dominant, recessive, additive . . .).

One can further estimate the attributable risk (AR) which describes the proportion of individuals in a population exhibiting a trait due to a given risk factor. This measure is important in quantitating the role of a specific factor in disease etiology and in terms of the public health impact of a risk factor. The public health relevance of this measure lies in estimating the proportion of cases of disease in the population that could be prevented if the exposure of interest were absent. AR is determined as follows:

AR=P _(E)(RR−1)/(P _(E)(RR−1)+1)

AR is the risk attributable to a biallelic marker allele or a biallelic marker haplotype. P_(E) is the frequency of exposure to an allele or a haplotype within the population at large; and RR is the relative risk which, is approximated with the odds ratio when the trait under study has a relatively low incidence in the general population.

IV.F. Identification Of Biallelic Markers In Linkage Disequilibrium With The Biallelic Markers of the Invention

Once a first biallelic marker has been identified in a genomic region of interest, the practitioner of ordinary skill in the art, using the teachings of the present invention, can easily identify additional biallelic markers in linkage disequilibrium with this first marker. As mentioned before any marker in linkage disequilibrium with a first marker associated with a trait will be associated with the trait. Therefore, once an association has been demonstrated between a given biallelic marker and a trait, the discovery of additional biallelic markers associated with this trait is of great interest in order to increase the density of biallelic markers in this particular region. The causal gene or mutation will be found in the vicinity of the marker or set of markers showing the highest correlation with the trait.

Identification of additional markers in linkage disequilibrium with a given marker involves: (a) amplifying a genomic fragment comprising a first biallelic marker from a plurality of individuals; (b) identifying of second biallelic markers in the genomic region harboring said first biallelic marker; (c) conducting a linkage disequilibrium analysis between said first biallelic marker and second biallelic markers; and (d) selecting said second biallelic markers as being in linkage disequilibrium with said first marker. Subcombinations comprising steps (b) and (c) are also contemplated.

Methods to identify biallelic markers and to conduct linkage disequilibrium analysis are described herein and can be carried out by the skilled person without undue experimentation. The present invention then also concerns biallelic markers which are in linkage disequilibrium with any of the specific biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908 and which are expected to present similar characteristics in terms of their respective association with a given trait.

Example 5 illustrates the measurement of linkage disequilibrium between a publicly known biallelic marker, the “ApoE Site A”, located within the Alzheimer's related ApoE gene, and other biallelic markers randomly derived from the genomic region containing the ApoE gene.

IV.G. Identification Of Functional Mutations

Once a positive association is confirmed with a biallelic marker of the present invention, the associated candidate gene can be scanned for mutations by comparing the sequences of a selected number of trait positive and trait negative individuals. In a preferred embodiment, functional regions such as exons and splice sites, promoters and other regulatory regions of the candidate gene are scanned for mutations. Preferably, trait positive individuals carry the haplotype shown to be associated with the trait and trait negative individuals do not carry the haplotype or allele associated with the trait. The mutation detection procedure is essentially similar to that used for biallelic site identification.

The method used to detect such mutations generally comprises the following steps: (a) amplification of a region of the candidate gene comprising a biallelic marker or a group of biallelic markers associated with the trait from DNA samples of trait positive patients and trait negative controls; (b) sequencing of the amplified region; (c) comparison of DNA sequences from trait-positive patients and trait-negative controls; and (d) determination of mutations specific to trait-positive patients. Subcombinations which comprise steps (b) and (c) are specifically contemplated.

It is preferred that candidate polymorphisms be then verified by screening a larger population of cases and controls by means of any genotyping procedure such as those described herein, preferably using a microsequencing technique in an individual test format. Polymorphisms are considered as candidate mutations when present in cases and controls at frequencies compatible with the expected association results.

V. Biallelic Markers Of The Invention In Methods Of Genetic Diagnostics

The biallelic markers of the present invention can also be used to develop diagnostics tests capable of identifying individuals who express a detectable trait as the result of a specific genotype or individuals whose genotype places them at risk of developing a detectable trait at a subsequent time. The trait analyzed using the present diagnostics may be any detectable trait, including a disease, a response to an agent acting on a disease, or side effects to an agent acting on a disease.

The diagnostic techniques of the present invention may employ a variety of methodologies to determine whether a test subject has a biallelic marker pattern associated with an increased risk of developing a detectable trait or whether the individual suffers from a detectable trait as a result of a particular mutation, including methods which enable the analysis of individual chromosomes for haplotyping, such as family studies, single sperm DNA analysis or somatic hybrids.

The present invention provides diagnostic methods to determine whether an individual is at risk of developing a disease or suffers from a disease resulting from a mutation or a polymorphism in a candidate gene of the present invention. The present invention also provides methods to determine whether an individual is likely to respond positively to an agent acting on a disease or whether an individual is at risk of developing an adverse side effect to an agent acting on a disease.

These methods involve obtaining a nucleic acid sample from the individual and, determining, whether the nucleic acid sample contains at least one allele or at least one biallelic marker haplotype, indicative of a risk of developing the trait or indicative that the individual expresses the trait as a result of possessing a particular candidate gene polymorphism or mutation (trait-causing allele).

Preferably, in such diagnostic methods, a nucleic acid sample is obtained from the individual and this sample is genotyped using methods described above in III. The diagnostics may be based on a single biallelic marker or a on group of biallelic markers.

In each of these methods, a nucleic acid sample is obtained from the test subject and the biallelic marker pattern of one or more of the biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, and 3735 to 3908 is determined.

In one embodiment, a PCR amplification is conducted on the nucleic acid sample to amplify regions in which polymorphisms associated with a detectable phenotype have been identified. The amplification products are sequenced to determine whether the individual possesses one or more polymorphisms associated with a detectable phenotype. The primers used to generate amplification products may comprise the primers of SEQ ID Nos. 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773. Alternatively, the nucleic acid sample is subjected to microsequencing reactions as described above to determine whether the individual possesses one or more polymorphisms associated with a detectable phenotype resulting from a mutation or a polymorphism in a candidate gene. In another embodiment, the nucleic acid sample is contacted with one or more allele specific oligonucleotide probes which, specifically hybridize to one or more candidate gene alleles associated with a detectable phenotype.

These diagnostic methods are extremely valuable as they can, in certain circumstances, be used to initiate preventive treatments or to allow an individual carrying a significant haplotype to foresee warning signs such as minor symptoms. In diseases in which attacks may be extremely violent and sometimes fatal if not treated on time, such as disease, the knowledge of a potential predisposition, even if this predisposition is not absolute, might contribute in a very significant manner to treatment efficacy. Similarly, a diagnosed predisposition to a potential side effect could immediately direct the physician toward a treatment for which such side effects have not been observed during clinical trials.

Diagnostics, which analyze and predict response to a drug or side effects to a drug, may be used to determine whether an individual should be treated with a particular drug. For example, if the diagnostic indicates a likelihood that an individual will respond positively to treatment with a particular drug, the drug may be administered to the individual. Conversely, if the diagnostic indicates that an individual is likely to respond negatively to treatment with a particular drug, an alternative course of treatment may be prescribed. A negative response may be defined as either the absence of an efficacious response or the presence of toxic side effects.

Clinical drug trials represent another application for the markers of the present invention. One or more markers indicative of response to an agent acting on a disease or to side effects to an agent acting on a disease may be identified using the methods described above. Thereafter, potential participants in clinical trials of such an agent may be screened to identify those individuals most likely to respond favorably to the drug and exclude those likely to experience side effects. In that way, the effectiveness of drug treatment may be measured in individuals who respond positively to the drug, without lowering the measurement as a result of the inclusion of individuals who are unlikely to respond positively in the study and without risking undesirable safety problems.

VI. Computer-Related Embodiments

In some embodiments of the present invention a computer to based system may support the on-line coordination between the identification of biallelic markers and the corresponding analysis of their frequency in the different groups.

As used herein the term “nucleic acid codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773” encompasses the nucleotide sequences of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773, fragments of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773, nucleotide sequences homologous to SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 or homologous to fragments of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773, and sequences complementary to all of the preceding sequences. As used herein the term “nucleic acid codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773” further encompasses the nucleotide sequences comprising, consisting essentially of, or consisting of any one of the following:

a) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 1 to 2260 or the complements thereof;

b) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 1 to 2260 or the complements thereof, further comprising the 1^(ST) allele of the polymorphic base of the respective SEQ ID number;

c) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 1 to 2260 or the complements thereof, further comprising the 2^(ND) allele of the polymorphic base of the respective SEQ ID number;

d) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 2261 to 3734 or the complements thereof;

e) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 2261 to 3734 or the complements thereof, further comprising the 1^(ST) allele of the polymorphic base of the respective SEQ ID number;

f) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 2261 to 3734 or the complements thereof, further comprising the 2^(ND) allele of the polymorphic base of the respective SEQ ID number;

g) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 3735 to 3908 or the complements thereof;

h) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 3735 to 3908 or the complements thereof, further comprising the 1^(ST) allele of the polymorphic base of the respective SEQ ID number;

i) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 3735 to 3908 or the complements thereof, further comprising the 2^(ND) allele of the polymorphic base of the respective SEQ ID number; and

j) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, or 21 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 or the complements thereof.

The “nucleic acid codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773” further encompass nucleotide sequences homologous to:

a) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 1 to 2260 or the complements thereof;

b) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 1 to 2260 or the complements thereof, further comprising the 1^(ST) allele of the polymorphic base of the respective SEQ ID number;

c) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 1 to 2260 or the complements thereof, further comprising the 2^(ND) allele of the polymorphic base of the respective SEQ ID number;

d) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 2261 to 3734 or the complements thereof;

e) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 2261 to 3734 or the complements thereof, further comprising the 1^(ST) allele of the polymorphic base of the respective SEQ ID number;

f) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 2261 to 3734 or the complements thereof, further comprising the 2^(ND) allele of the polymorphic base of the respective SEQ ID number;

g) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 3735 to 3908 or the complements thereof;

h) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 3735 to 3908 or the complements thereof, further comprising the 1^(ST) allele of the polymorphic base of the respective SEQ ID number;

i) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, 22, 23, 24, 25, 30, 35, 43, 44, 45, 46 or 47 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 3735 to 3908 or the complements thereof, further comprising the 2^(ND) allele of the polymorphic base of the respective SEQ ID number; and

j) a contiguous span of at least 8, 10, 12, 15, 18, 19, 20, or 21 nucleotides, to the extent that a contiguous span of these lengths is consistent with the lengths of the particular Sequence ID, of any of SEQ ID Nos. 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 or the complements thereof.

Homologous sequences refer to a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% homology to these contiguous spans. Homology may be determined using any method described herein, including BLAST2N with the default parameters or with any modified parameters. Homologous sequences also may include RNA sequences in which uridines replace the thymines in the nucleic acid codes of the invention. It will be appreciated that the nucleic acid codes of the invention can be represented in the traditional single character format (See the inside back cover of Stryer, Lubert. Biochemistry, 3^(rd) edition. W. H Freeman & Co., New York.) or in any other format or code which records the identity of the nucleotides in a sequence.

It should be noted that the nucleic acid codes of the invention further encompass all of the polynucleotides disclosed, described or claimed in the present application. Moveover, the present invention specifically contemplates computer readable media and computer systems wherein such codes are stored individually or in any combination.

It will be appreciated by those skilled in the art that the nucleic acid codes of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. As used herein, the words “recorded” and “stored” refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any of the presently known methods for recording information on a computer readable medium to generate embodiments comprising one or more of the nucleic acid codes of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773. A particularly preferred embodiment of the present invention is a computer readable medium having recorded thereon at least 2, 5, 10, 15, 20, 25, 30, 50, 100, 200, 500, 1000, 2000, or 5000 nucleic acid codes of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773.

Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media. For example, the computer readable media may be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.

Embodiments of the present invention include systems, particularly computer systems which store and manipulate the sequence information described herein. One example of a computer system 100 is illustrated in block diagram form in FIG. 14. As used herein, “a computer system” refers to the hardware components, software components, and data storage components used to analyze the nucleotide sequences of the nucleic acid codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773. In one embodiment, the computer system 100 is a Sun Enterprise 1000 server (Sun Microsystems, Palo Alto, Calif.). The computer system 100 preferably includes a processor for processing, accessing and manipulating the sequence data. The processor 105 can be any well-known type of central processing unit, such as the Pentium III from Intel Corporation, or similar processor from Sun, Motorola, Compaq or International Business Machines.

Preferably, the computer system 100 is a general purpose system that comprises the processor 105 and one or more internal data storage components 110 for storing data, and one or more data retrieving devices for retrieving the data stored on the data storage components. A skilled artisan can readily appreciate that any one of the currently available computer systems are suitable.

In one particular embodiment, the computer system 100 includes a processor 105 connected to a bus which is connected to a main memory 115 (preferably implemented as RAM) and one or more internal data storage devices 110, such as a hard drive and/or other computer readable media having data recorded thereon. In some embodiments, the computer system 100 further includes one or more data retrieving device 118 for reading the data stored on the internal data storage devices 110.

The data retrieving device 118 may represent, for example, a floppy disk drive, a compact disk drive, a magnetic tape drive, etc. In some embodiments, the internal data storage device 110 is a removable computer readable medium such as a floppy disk, a compact disk, a magnetic tape, etc. containing control logic and/or data recorded thereon. The computer system 100 may advantageously include or be programmed by appropriate software for reading the control logic and/or the data from the data storage component once inserted in the data retrieving device.

The computer system 100 includes a display 120 which is used to display output to a computer user. It should also be noted that the computer system 100 can be linked to other computer systems 125 a-c in a network or wide area network to provide centralized access to the computer system 100.

Software for accessing and processing the nucleotide sequences of the nucleic acid codes of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 (such as search tools, compare tools, and modeling tools etc.) may reside in main memory 115 during execution.

In some embodiments, the computer system 100 may further comprise a sequence comparer for comparing the above-described nucleic acid codes of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 stored on a computer readable medium to reference nucleotide or polypeptide sequences stored on a computer readable medium. A “sequence comparer” refers to one or more programs which are implemented on the computer system 100 to compare a nucleotide sequence with other nucleotide sequences and/or compounds stored within the data storage means. For example, the sequence comparer may compare the nucleotide sequences of the nucleic acid codes of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 stored on a computer readable medium to reference sequences stored on a computer readable medium to identify homologies or structural motifs. The various sequence comparer programs identified elsewhere in this patent specification are particularly contemplated for use in this aspect of the invention.

FIG. 15 is a flow diagram illustrating one embodiment of a process 200 for comparing a new nucleotide or protein sequence with a database of sequences in order to determine the homology levels between the new sequence and the sequences in the database. The database of sequences can be a private database stored within the computer system 100, or a public database such as GENBANK that is available through the Internet.

The process 200 begins at a start state 201 and then moves to a state 202 wherein the new sequence to be compared is stored to a memory in a computer system 100. As discussed above, the memory could be any type of memory, including RAM or an internal storage device.

The process 200 then moves to a state 204 wherein a database of sequences is opened for analysis and comparison. The process 200 then moves to a state 206 wherein the first sequence stored in the database is read into a memory on the computer. A comparison is then performed at a state 210 to determine if the first sequence is the same as the second sequence. It is important to note that this step is not limited to performing an exact comparison between the new sequence and the first sequence in the database. Well-known methods are known to those of skill in the art for comparing two nucleotide or protein sequences, even if they are not identical. For example, gaps can be introduced into one sequence in order to raise the homology level between the two tested sequences. The parameters that control whether gaps or other features are introduced into a sequence during comparison are normally entered by the user of the computer system.

Once a comparison of the two sequences has been performed at the state 210, a determination is made at a decision state 210 whether the two sequences are the same. Of course, the term “same” is not limited to sequences that are absolutely identical. Sequences that are within the homology parameters entered by the user will be marked as “same” in the process 200.

If a determination is made that the two sequences are the same, the process 200 moves to a state 214 wherein the name of the sequence from the database is displayed to the user. This state notifies the user that the sequence with the displayed name fulfills the homology constraints that were entered. Once the name of the stored sequence is displayed to the user, the process 200 moves to a decision state 218 wherein a determination is made whether more sequences exist in the database. If no more sequences exist in the database, then the process 200 terminates at an end state 220. However, if more sequences do exist in the database, then the process 200 moves to a state 224 wherein a pointer is moved to the next sequence in the database so that it can be compared to the new sequence. In this manner, the new sequence is aligned and compared with every sequence in the database.

It should be noted that if a determination had been made at the decision state 212 that the sequences were not homologous, then the process 200 would move immediately to the decision state 218 in order to determine if any other sequences were available in the database for comparison.

Accordingly, one aspect of the present invention is a computer system comprising a processor, a data storage device having stored thereon a nucleic acid code of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773, a data storage device having retrievably stored thereon reference nucleotide sequences or polypeptide sequences to be compared to the nucleic acid code of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 and a sequence comparer for conducting the comparison. The sequence comparer may indicate a homology level between the sequences compared or identify structural motifs in the above described nucleic acid code of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 or it may identify structural motifs in sequences which are compared to these nucleic acid codes and polypeptide codes. In some embodiments, the data storage device may have stored thereon the sequences of at least 2, 5, 10, 15, 20, 25, 30, 50, 100, 200, 500, 1000, 2000, or 5000 of the nucleic acid codes of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773.

Another aspect of the present invention is a method for determining the level of homology between a nucleic acid code of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 and a reference nucleotide sequence, comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through the use of a computer program which determines homology levels and determining homology between the nucleic acid code and the reference nucleotide sequence with the computer program. The computer program may be any of a number of computer programs for determining homology levels, including those specifically enumerated herein, including BLAST2N with the default parameters or with any modified parameters. The method may be implemented using the computer systems described above. The method may also be performed by reading at least 2, 5, 10, 15, 20, 25, 30, 50, 100, 200, 500, 1000, 2000, or 5000 of the above described nucleic acid codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 through use of the computer program and determining homology between the nucleic acid codes and reference nucleotide sequences.

FIG. 16 is a flow diagram illustrating one embodiment of a process 250 in a computer for determining whether two sequences are homologous. The process 250 begins at a start state 252 and then moves to a state 254 wherein a first sequence to be compared is stored to a memory. The second sequence to be compared is then stored to a memory at a state 256. The process 250 then moves to a state 260 wherein the first character in the first sequence is read and then to a state 262 wherein the first character of the second sequence is read. It should be understood that if the sequence is a nucleotide sequence, then the character would normally be either A, T, C, G or U. If the sequence is a protein sequence, then it should be in the single letter amino acid code so that the first and sequence sequences can be easily compared.

A determination is then made at a decision state 264 whether the two characters are the same. If they are the same, then the process 250 moves to a state 268 wherein the next characters in the first and second sequences are read. A determination is then made whether the next characters are the same. If they are, then the process 250 continues this loop until two characters are not the same. If a determination is made that the next two characters are not the same, the process 250 moves to a decision state 274 to determine whether there are any more characters either sequence to read.

If there aren't any more characters to read, then the process 250 moves to a state 276 wherein the level of homology between the first and second sequences is displayed to the user. The level of homology is determined by calculating the proportion of characters between the sequences that were the same out of the total number of sequences in the first sequence. Thus, if every character in a first 100 nucleotide sequence aligned with a every character in a second sequence, the homology level would be 100%.

Alternatively, the computer program may be a computer program which compares the nucleotide sequences of the nucleic acid codes of the present invention, to reference nucleotide sequences in order to determine whether the nucleic acid code of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 differs from a reference nucleic acid sequence at one or more positions. Optionally such a program records the length and identity of inserted, deleted or substituted nucleotides with respect to the sequence of either the reference polynucleotide or the nucleic acid code of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773. In one embodiment, the computer program may be a program which determines whether the nucleotide sequences of the nucleic acid codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 contain a biallelic marker or single nucleotide polymorphism (SNP) with respect to a reference nucleotide sequence. This single nucleotide polymorphism may comprise a single base substitution, insertion, or deletion, while this biallelic marker may comprise about one to ten consecutive bases substituted, inserted or deleted.

Accordingly, another aspect of the present invention is a method for determining whether a nucleic acid code of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 differs at one or more nucleotides from a reference nucleotide sequence comprising the steps of reading the nucleic acid code and the reference nucleotide sequence through use of a computer program which identifies differences between nucleic acid sequences and identifying differences between the nucleic acid code and the reference nucleotide sequence with the computer program. In some embodiments, the computer program is a program which identifies single nucleotide polymorphisms. The method may be implemented by the computer systems described above and the method illustrated in FIG. 16. The method may also be performed by reading at least 2, 5, 10, 15, 20, 25, 30, 50, 100, 200, 500, 1000, 2000, or 5000 of the nucleic acid codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 and the reference nucleotide sequences through the use of the computer program and identifying differences between the nucleic acid codes and the reference nucleotide sequences with the computer program. In other embodiments the computer based system may further comprise an identifier for identifying features within the nucleotide sequences of the nucleic acid codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773.

An “identifier” refers to one or more programs which identifies certain features within the above-described nucleotide sequences of the nucleic acid codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773. In one embodiment, the identifier may comprise a program which identifies an open reading frame in the cDNAs codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773.

FIG. 17 is a flow diagram illustrating one embodiment of an identifier process 300 for detecting the presence of a feature in a sequence. The process 300 begins at a start state 302 and then moves to a state 304 wherein a first sequence that is to be checked for features is stored to a memory 115 in the computer system 100. The process 300 then moves to a state 306 wherein a database of sequence features is opened. Such a database would include a list of each feature's attributes along with the name of the feature. For example, a feature name could be “Initiation Codon” and the attribute would be “ATG”. Another example would be the feature name “TAATAA Box” and the feature attribute would be “TAATAA”. An example of such a database is produced by the University of Wisconsin Genetics Computer Group (www.gcg.com).

Once the database of features is opened at the state 306, the process 300 moves to a state 308 wherein the first feature is read from the database. A comparison of the attribute of the first feature with the first sequence is then made at a state 310. A determination is then made at a decision state 316 whether the attribute of the feature was found in the first sequence. If the attribute was found, then the process 300 moves to a state 318 wherein the name of the found feature is displayed to the user.

The process 300 then moves to a decision state 320 wherein a determination is made whether move features exist in the database. If no more features do exist, then the process 300 terminates at an end state 324. However, if more features do exist in the database, then the process 300 reads the next sequence feature at a state 326 and loops back to the state 310 wherein the attribute of the next feature is compared against the first sequence.

It should be noted, that if the feature attribute is not found in the first sequence at the decision state 316, the process 300 moves directly to the decision state 320 in order to determine if any more features exist in the database.

Accordingly, another aspect of the present invention is a method of identifying a feature within the nucleic acid codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 comprising reading the nucleic acid code(s) through the use of a computer program which identifies features therein and identifying features within the nucleic acid code(s) with the computer program. In one embodiment, computer program comprises a computer program which identifies open reading frames. The method may be performed by reading a single sequence or at least 2, 5, 10, 15, 20, 25, 30, 50, 100, 200, 500, 1000, 2000, or 5000 of the nucleic acid codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 through the use of the computer program and identifying features within the nucleic acid codes with the computer program.

The nucleic acid codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 may be stored and manipulated in a variety of data processor programs in a variety of formats. For example, the nucleic acid codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 may be stored as text in a word processing file, such as MicrosoftWORD or WORDPERFECT or as an ASCII file in a variety of database programs familiar to those of skill in the art, such as DB2, SYBASE, or ORACLE. In addition, many computer programs and databases may be used as sequence comparers, identifiers, or sources of reference nucleotide sequences to be compared to the nucleic acid codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773. The following list is intended not to limit the invention but to provide guidance to programs and databases which are useful with the nucleic acid codes of SEQ ID NOs. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908, 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773.

The programs and databases which may be used include, but are not limited to: MacPattern (EMBL), DiscoveryBase (Molecular Applications Group), GeneMine (Molecular Applications Group), Look (Molecular Applications Group), MacLook (Molecular Applications Group), BLAST and BLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215: 403 (1990)), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85: 2444 (1988)), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-245, 1990), Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (Molecular Simulations Inc.), Cerius².DBAccess (Molecular Simulations Inc.), HypoGen (Molecular Simulations Inc.), Insight II, (Molecular Simulations Inc.), Discover (Molecular Simulations Inc.), CHARMm (Molecular Simulations Inc.), Felix (Molecular Simulations Inc.), DelPhi, (Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.), Homology (Molecular Simulations Inc.), Modeler (Molecular Simulations Inc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design (Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.), WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer (Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), the MDL Available Chemicals Directory database, the MDL Drug Data Report data base, the Comprehensive Medicinal Chemistry database, Derwents's World Drug Index database, the BioByteMasterFile database, the Genbank database, and the Genseqn database. Many other programs and data bases would be apparent to one of skill in the art given the present disclosure.

Motifs which may be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.

It should be noted that the nucleic acid codes of the invention further encompass all of the polynucleotides disclosed, described or claimed in the present application. Moreover, the present invention specifically contemplates the storage of such codes on computer readable media and computer systems individually or in any combination, as well as the use of such codes and combinations in the methods of VI.

VII. Mapping and Maps Comprising the Biallelic Markers of the Invention

The human haploid genome contains an estimated 80,000 to 100,000 or more genes scattered on a 3×10⁹ base-long double stranded DNA shared among the 24 chromosomes. Each human being is diploid, i.e. possesses two haploid genomes, one from paternal origin, the other from maternal origin. The sequence of the human genome varies among individuals in a population About 10⁷ sites scattered along the 3×10⁹ base pairs of DNA are polymorphic, existing in at least two variant forms called alleles. Most of these polymorphic sites are generated by single base substitution mutations and are biallelic. Less than 10⁵ polymorphic sites are due to more complex changes and are very often multi-allelic, i.e. exist in more than two allelic forms. At a given polymorphic site, any individual (diploid), can be either homozygous (twice the same allele) or heterozygous (two different alleles). A given polymorphism or rare mutation can be either neutral (no effect on trait), or functional, i.e. responsible for a particular genetic trait.

Genetic Maps

The first step towards the identification of genes associated with a detectable trait, such as a disease or any other detectable trait, consists in the localization of genomic regions containing trait-causing genes using genetic mapping methods. The preferred traits contemplated within the present invention relate to fields of therapeutic interest; in particular embodiments, they will be disease traits and/or drug response traits, reflecting drug efficacy or toxicity. Traits can either be “binary”, e.g. diabetic vs. non diabetic, or “quantitative”, e.g. elevated blood pressure. Individuals affected by a quantitative trait can be classified according to an appropriate scale of trait values, e.g. blood pressure ranges. Each trait value range can then be analyzed as a binary trait. Patients showing a trait value within one such range will be studied in comparison with patients showing a trait value outside of this range. In such a case, genetic analysis methods will be applied to subpopulations of individuals showing trait values within defined ranges.

Genetic mapping involves the analysis of the segregation of polymorphic loci in trait positive and trait-negative populations. Polymorphic loci constitute a small fraction of the human genome (less than 1%), compared to the vast majority of human genomic DNA which is identical in sequence among the chromosomes of different individuals. Among all existing human polymorphic loci, genetic markers can be defined as genome-derived polynucleotides which are sufficiently polymorphic to allow a reasonable probability that a randomly selected person will be heterozygous, and thus informative for genetic analysis by methods such as linkage analysis or association studies.

A genetic map consists of a collection of polymorphic markers which have been positioned on the human chromosomes. Genetic maps may be combined with physical maps, collections of ordered overlapping fragments of genomic DNA whose arrangement along the human chromosomes is known. The optimal genetic map should possess the following characteristics:

the density of the genetic markers scattered along the genome should be sufficient to allow the identification and localization of any trait-related polymorphism,

each marker should have an adequate level of heterozygosity, so as to be informative in a large percentage of different meioses,

all markers should be easily typed on a routine basis, at a reasonable expense, and in a reasonable amount of time,

the entire set of markers per chromosome should be ordered in a highly reliable fashion.

However, while the above maps are optimal, it will be appreciated that the maps of the present invention may be used in the individual marker and haplotype association analyses described below without the necessity of determining the order of biallelic markers derived from a single BAC with respect to one another.

Construction of a Physical Map

The first step in constructing a high density genetic map of biallelic markers is the construction of a physical map. Physical maps consist of ordered, overlapping cloned fragments of genomic DNA covering a portion of the genome, preferably covering one or all chromosomes. Obtaining a physical map of the genome entails constructing and ordering a genomic DNA library. For an example of a complete explanation of the construction of a physical map from a BAC library see related PCT Application No. PCT/IB98/00193 filed Jul. 17, 1998, the disclosure of which is incorporated herein by reference in its entirety. The methods disclosed therein can be used to generate larger more complete sets of markers and entire maps of the human genome comprising the map-relate biallelic markers of the invention.

Biallelic Markers

It will be appreciated that the ordered DNA fragments containing these groups of biallelic markers need not completely cover the genomic regions of these lengths but may instead be incomplete contigs having one or more gaps therein. As discussed in further detail below, biallelic markers may be used in single maker and haplotype association analyses regardless of the completeness of the corresponding physical contig harboring them.

Using the procedures above, 3908 biallelic markers, each having two alleles, were identified using sequences obtained from BACs which had been localized on the genome. In some cases, markers were identified using pooled BACs and thereafter reassigned to individual BACs using STS screening procedures such as those described in Examples 1 and 2. The sequences of these biallelic markers are provided in the accompanying Sequence Listing as SEQ ID Nos. 1 to 3908. Although the sequences of SEQ ID Nos. 1 to 3908 will be used as exemplary markers throughout the present application, these markers are not limited to markers having the exact flanking sequences surrounding the polymorphic bases which are enumerated in SEQ ID Nos. 1 to 3908. Rather, it will be appreciated that the flanking sequences surrounding the polymorphic bases of SEQ ID Nos. 1 to 3908 may be lengthened or shortened to any extent compatible with their intended use and the present invention specifically contemplates such sequences. The sequences of these biallelic markers may be used to construct genomic maps as well as in the gene identification and diagnostic techniques described herein. It will be appreciated that the biallelic markers referred to herein may be of any length compatible with their intended use provided that the markers include the polymorphic base, and the present invention specifically contemplates such sequences.

Some of the markers of SEQ ID Nos: 1 to 3908 as well as related amplification and microsequencing primers were disclosed in the instant priority documents. However, some of the earlier described amplification primers and microsequencing primers did not have the precise sequence lengths disclosed in the instant application. It will be appreciated that either length of primers may be used in the methods disclosed in the present application.

In addition, the internal identification numbers used to identify the biallelic markers disclosed in U.S. Provisional Patent Application Serial No. 60/082,614 filed Apr. 21, 1998 have been revised to include additional numbers on the end. For example, the marker formerly given the internal identification number 99-1091 was given the revised internal identification number 99-1091-446. Therefore, it will be appreciated that shortened identification numbers and extended identification numbers which overlap one another refer to the same markers.

Ordering of Biallelic Markers

Biallelic markers can be ordered to determine their positions along chromosomes, preferably subchromosomal regions, by methods known in the art as well as those disclosed in PCT Application No. PCT/IB98/00193 filed Jul. 17, 1998, and U.S. Provisional Patent Application Serial No. 60/082,614 filed Apr. 21, 1998.

The positions of the biallelic markers along chromosomes may be determined using a variety of methodologies. In one approach, radiation hybrid mapping is used. Radiation hybrid (RH) mapping is a somatic cell genetic approach that can be used for high resolution mapping of the human genome. In this approach, cell lines containing one or more human chromosomes are lethally irradiated, breaking each chromosome into fragments whose size depends on the radiation dose. These fragments are rescued by fusion with cultured rodent cells, yielding subclones containing different portions of the human genome. This technique is described by Benham et al. (Genomics 4:509-517, 1989) and Cox et al., (Science 250:245-250, 1990), the entire contents of which are hereby incorporated by reference. The random and independent nature of the subclones permits efficient mapping of any human genome marker. Human DNA isolated from a panel of 80-100 cell lines provides a mapping reagent for ordering biallelic markers. In this approach, the frequency of breakage between markers is used to measure distance, allowing construction of fine resolution maps as has been done for ESTs (Schuler et al., Science 274:540-546, 1996, hereby incorporated herein by reference in its entirety).

RH mapping has been used to generate a high-resolution whole genome radiation hybrid map of human chromosome 17q22-q25.3 across the genes for growth hormone (GH) and thymidine kinase (TK) (Foster et al., Genomics 33:185-192, 1996), the region surrounding the Gorlin syndrome gene (Obermayr et al., Eur. J. Hum. Genet. 4:242-245, 1996), 60 loci covering the entire short arm of chromosome 12 (Raeymaekers et al., Genomics 29:170-178, 1995), the region of human chromosome 22 containing the neurofibromatosis type 2 locus (Frazer et al., Genomics 14:574-584, 1992) and 13 loci on the long arm of chromosome 5 (Warrington et al., Genomics 11:701-708, 1991, the disclosure of which is hereby incorporated herein by reference in its entirety).

Alternatively, PCR based techniques and human-rodent somatic cell hybrids may be used to determine the positions of the biallelic markers on the chromosomes. In such approaches, oligonucleotide primer pairs which are capable of generating amplification products containing the polymorphic bases of the biallelic markers are designed. Preferably, the oligonucleotide primers are 18-23 bp in length and are designed for PCR amplification. The creation of PCR primers from known sequences is well known to those with skill in the art. For a review of PCR technology see Erlich, H. A., PCR Technology: Principles and Applications for DNA Amplification. 1992. W. H. Freeman and Co., New York, the disclosure of which is hereby incorporated herein by reference in its entirety.

The primers are used in polymerase chain reactions (PCR) to amplify templates from total human genomic DNA. PCR conditions are as follows: 60 ng of genomic DNA is used as a template for PCR with 80 ng of each oligonucleotide primer, 0.6 unit of Taq polymerase, and 1 mCu of a ³²P-labeled deoxycytidine triphosphate. The PCR is performed in a microplate thermocycler (Techne) under the following conditions: 30 cycles of 94° C., 1.4 min; 55° C., 2 min; and 72° C., 2 min; with a final extension at 72° C. for 10 min. The amplified products are analyzed on a 6% polyacrylamide sequencing gel and visualized by autoradiography. If the length of the resulting PCR product is identical to the length expected for an amplification product containing the polymorphic base of the biallelic marker, then the PCR reaction is repeated with DNA templates from two panels of human-rodent somatic cell hybrids, BIOS PCRable DNA (BIOS Corporation) and NIGMS Human-Rodent Somatic Cell Hybrid Mapping Panel Number 1 (NIGMS, Camden, N.J.).

PCR is used to screen a series of somatic cell hybrid cell lines containing defined sets of human chromosomes for the presence of a given biallelic marker. DNA is isolated from the somatic hybrids and used as starting templates for PCR reactions using the primer pairs from the biallelic marker. Only those somatic cell hybrids with chromosomes containing the human sequence corresponding to the biallelic marker will yield an amplified fragment. The biallelic markers are assigned to a chromosome by analysis of the segregation pattern of PCR products from the somatic hybrid DNA templates. The single human chromosome present in all cell hybrids that give rise to an amplified fragment is the chromosome containing that biallelic marker. For a review of techniques and analysis of results from somatic cell gene mapping experiments. (See Ledbetter et al., Genomics 6:475-481 (1990 the disclosure of which is incorporated herein by reference in its entirety).)

Example 2 describes a preferred method for positioning of biallelic markers on clones, such as BAC clones, obtained from genomic DNA libraries. Using such procedures, a number of BAC clones carrying selected biallelic markers can be isolated. The position of these BAC clones on the human genome can be defined by performing STS screening as described in Example 1. Preferably, to decrease the number of STSs to be tested, each BAC can be localized on chromosomal or subchromosomal regions by procedures such as those described in Examples 3 and 4. This localization will allow the selection of a subset of STSs corresponding to the identified chromosomal or subchromosomal region. Testing each BAC with such a subset of STSs and taking account of the position and order of the STSs along the genome will allow a refined positioning of the corresponding biallelic marker along the genome.

In other embodiments, if the DNA library used to isolate BAC inserts or any type of genomic DNA fragments harboring the selected biallelic markers already constitute a physical map of the genome or any portion thereof, using the known order of the DNA fragments will allow the order of the biallelic markers to be established.

As discussed above, it will be appreciated that markers carried by the same fragment of genomic DNA, such as the insert in a BAC clone, need not necessarily be ordered with respect to one another within the genomic fragment to conduct single point or haplotype association analyses. However, in other embodiments of the present maps, the order of biallelic markers carried by the same fragment of genomic DNA may be determined.

The positions of the biallelic markers used to construct the maps of the present invention, including the map-related biallelic markers of the invention, may be assigned to subchromosomal locations using Fluorescence In Situ Hybridization (FISH) (Cherif et al., Proc. Natl. Acad. Sci. U.S.A., 87:6639-6643 (1990), the disclosure of which is incorporated herein by reference in its entirety). FISH analysis is described in Example 3.

The ordering analyses may be conducted to generate an integrated genome wide genetic map comprising about 20,000, 40,000, 60,000, 80,000, 100,000, 120,000 biallelic markers with a roughly consistent number of biallelic marker per BAC. In some embodiments, the map includes one or more markers selected from the group consisting of the sequences of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto.

Alternatively, maps having the above-specified average numbers of biallelic markers per BAC which comprise smaller portions of the genome, such as a set of chromosomes, a single chromosome, a particular subchromosomal region, or any other desired portion of the genome, may also be constructed using the procedures provided herein.

In some embodiments, the biallelic markers in the map are separated from one another by an average distance of 10-200 kb, 15-150 kb, 20-100 kb, 100-150 kb, 50-100 kb, or 25-50 kb. Maps having the above-specified intermarker distances which comprise smaller portions of the genome, such as a set of chromosomes, a single chromosome, a particular subchromosomal region, or any other desired portion of the genome, may also be constructed using the procedures provided herein.

FIG. 2, showing the results of computer simulations of the distribution of inter-marker spacing on a randomly distributed set of biallelic markers, indicates the percentage of biallelic markers which will be spaced a given distance apart for a given number of markers/BAC in the genomic map (assuming 20,000 BACs constituting a minimally overlapping array covering the entire genome are evaluated). One hundred iterations were performed for each simulation (20,000 marker map, 40,000 marker map, 60,000 marker map, 120,000 marker map).

As illustrated in FIG. 2a, 98% of inter-marker distances will be lower than 150 kb provided 60,000 evenly distributed markers are generated (3 per BAC); 90% of inter-marker distances will be lower than 150 kb provided 40,000 evenly distributed markers are generated (2 per BAC); and 50% of inter-marker distances will be lower than 150 kb provided 20,000 evenly distributed markers are generated (1 per BAC).

As illustrated in FIG. 2b, 98% of inter-marker distances will be lower than 80 kb provided 120,000 evenly distributed markers are generated (6 per BAC); 80% of inter-marker distances will be lower than 80 kb provided 60,000 evenly distributed markers are generated (3 per BAC); and 15% of inter-marker distances will be lower than 80 kb provided 20,000 evenly distributed markers are generated (1 per BAC).

As already mentioned, high density biallelic marker maps allow association studies to be performed to identify genes involved in complex traits.

Tables 9 to 11 provide the genomic location of biallelic markers described herein. Listed are chromosomal regions and subregions to which biallelic markers were assigned using the methods of Example 3 and by screening BAC sequences against published and unpublished STSs.

In particular, the locations of markers listed in table 9 are locations for which adjacent STSs are publicly available. The column “adjacent STS” provides the public accession numbers of STSs localised on the same BAC with the subject biallelic marker as well as aliases for said STSs. As noted above, all of the marker localisations provided in Table 9 are confirmed by fluorescence in situ hybridization methods and public STS screening.

Table 10 describes chromosomal locations for biallelic markers for which no public adjacent STSs were available. Thus, Table 10 provides biallelic markers for which chromosomal localisations obtained by methods of FISH were confirmed by unpublished STSs, localisations which were obtained only by FISH, and localisations obtained by FISH which were discordant with localisations obtained from unpublished STSs.

Biallelic markers for which localisation were unconfirmed due to discordant localisation from STS screening and FISH methods are further provided in Table 11. The 204, 205, 225, 273, 274, 1723, 1732, 1743 localisations of these biallelic markers listed in Table 11 are those obtained by FISH methods, and may thus be considered as potential localisations. Table 11 includes certain markers also listed in Table 10.

Linkage Disequilibrium

The present invention then also concerns biallelic markers in linkage disequilibrium with the specific biallelic markers described above and which are expected to present similar characteristics in terms of their respective association with a given trait. In a preferred embodiment, the present invention concerns the biallelic markers that are in linkage disequilibrium with the biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto.

LD among a set of biallelic markers having an adequate heterozygosity rate can be determined by genotyping between 50 and 1000 unrelated individuals, preferably between 75 and 200, more preferably around 100. Genotyping a biallelic marker consists of determining the specific allele carried by an individual at the given polymorphic base of the biallelic marker. Genotyping can be performed using similar methods as those described above for the generation of the biallelic markers, or using other genotyping methods such as those further described below.

Genome-wide linkage disequilibrium mapping aims at identifying, for any trait-causing allele being searched, at least one biallelic marker in linkage disequilibrium with said trait-causing allele. Preferably, in order to enhance the power of linkage disequilibrium maps, in some embodiments, the biallelic markers therein have average inter-marker distances of 150 kb or less, 75 kb or less, or 50 kb or less, 30 kb or less, or 25 kb or less to accommodate the fact that, in some regions of the genome, the detection of linkage disequilibrium requires lower inter-marker distances.

The present invention provides methods to generate biallelic marker maps with average inter-marker distances of 150 kb or less. In some embodiments, the mean distance between biallelic markers constituting the high density map will be less than 75 kb, preferably less than 50 kb. Further preferred maps according to the present invention contain markers that are less than 37.5 kb apart. In highly preferred embodiments, the average inter-marker spacing for the biallelic markers constituting very high density maps is less than 30 kb, most preferably less than 25 kb.

Genetic maps containing biallelic markers (including the biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto) may be used to identify and isolate genes associated with detectable traits. The use of the genetic maps of the present invention is described in more detail below.

VIII. Use of HIigh Density Biallelic Marker Maps to Identify Genes Associated with Detectable Traits

One embodiment of the present invention comprises methods for identifying and isolating genes associated with a detectable trait using the biallelic marker maps of the present invention.

In the past, the identification of genes linked with detectable traits has relied on a statistical approach called linkage analysis. Linkage analysis is based upon establishing a correlation between the transmission of genetic markers and that of a specific trait throughout generations within a family. In this approach, all members of a series of affected families are genotyped with a few hundred markers, typically microsatellite markers, which are distributed at an average density of one every 10 Mb. By comparing genotypes in all family members, one can attribute sets of alleles to parental haploid genomes (haplotyping or phase determination). The origin of recombined fragments is then determined in the offspring of all families. Those that co-segregate with the trait are tracked. After pooling data from all families, statistical methods are used to determine the likelihood that the marker and the trait are segregating independently in all families. As a result of the statistical analysis, one or several regions having a high probability of harboring a gene linked to the trait are selected as candidates for further analysis. The result of linkage analysis is considered as significant (i.e. there is a high probability that the region contains a gene involved in a detectable trait) when the chance of independent segregation of the marker and the trait is lower than 1 in 1000 (expressed as a LOD score>3). Generally, the length of the candidate region identified using linkage analysis is between 2 and 20 Mb.

Once a candidate region is identified as described above, analysis of recombinant individuals using additional markers allows further delineation of the candidate linked region.

Linkage analysis studies have generally relied on the use of a maximum of 5,000 microsatellite markers, thus limiting the maximum theoretical attainable resolution of linkage analysis to ca. 600 kb on average.

Linkage analysis has been successfully applied to map simple genetic traits that show clear Mendelian inheritance patterns and which have a high penetrance (penetrance is the ratio between the number of trait-positive carriers of allele a and the total number of a carriers in the population). About 100 pathological trait-causing genes were discovered using linkage analysis over the last 10 years. In most of these cases, the majority of affected individuals had affected relatives and the detectable trait was rare in the general population (frequencies less than 0.1%). In about 10 cases, such as Alzheimer's Disease, breast cancer, and Type II diabetes, the detectable trait was more common but the allele associated with the detectable trait was rare in the affected population. Thus, the alleles associated with these traits were not responsible for the trait in all sporadic cases.

Linkage analysis suffers from a variety of drawbacks. First, linkage analysis is limited by its reliance on the choice of a genetic model suitable for each studied trait. Furthermore, as already mentioned, the resolution attainable using linkage analysis is limited, and complementary studies are required to refine the analysis of the typical 2 Mb to 20 Mb regions initially identified through linkage analysis.

In addition, linkage analysis approaches have proven difficult when applied to complex genetic traits, such as those due to the combined action of multiple genes and/or environmental factors. In such cases, too large an effort and cost are needed to recruit the adequate number of affected families required for applying linkage analysis to these situations, as recently discussed by Risch, N. and Merikangas, K. (Science 273:1516-1517 (1996), the disclosure of which is incorporated herein by reference in its entirety).

Finally, linkage analysis cannot be applied to the study of traits for which no large informative families are available. Typically, this will be the case in any attempt to identify trait-causing alleles involved in sporadic cases, such as alleles associated with positive or negative responses to drug treatment.

The present genetic maps and biallelic markers (including the biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto) may be used to identify and isolate genes associated with detectable traits using association studies, an approach which does not require the use of affected families and which permits the identification of genes associated with sporadic traits.

Association Studies

As already mentioned, any gene responsible or partly responsible for a given trait will be in linkage disequilibrium with some flanking markers. To map such a gene, specific alleles of these flanking markers which are associated with the gene or genes responsible for the trait are identified. Although the following discussion of techniques for finding the gene or genes associated with a particular trait using linkage disequilibrium mapping, refers to locating a single gene which is responsible for the trait, it will be appreciated that the same techniques may also be used to identify genes which are partially responsible for the trait.

Association studies may be conducted within the general population (as opposed to the linkage analysis techniques discussed above which are limited to studies performed on related individuals in one or several affected families).

Association between a biallelic marker A and a trait T may primarily occur as a result of three possible relationships between the biallelic marker and the trait.

First, allele a of biallelic marker A may be directly responsible for trait T (e.g., Apo E ε 4 site A and Alzheimer's disease). However, since the majority of the biallelic markers used in genetic mapping studies are selected randomly, they mainly map outside of genes. Thus, the likelihood of allele a being a functional mutation directly related to trait T is very low.

Second, an association between a biallelic marker A and a trait T may also occur when the biallelic marker is very closely linked to the trait locus. In other words, an association occurs when allele a is in linkage disequilibrium with the trait-causing allele. When the biallelic marker is in close proximity to a gene responsible for the trait, more extensive genetic mapping will ultimately allow a gene to be discovered near the marker locus which carries mutations in people with trait T (i.e. the gene responsible for the trait or one of the genes responsible for the trait). As will be further exemplified below, using a group of biallelic markers which are in close proximity to the gene responsible for the trait the location of the causal gene can be deduced from the profile of the association curve between the biallelic markers and the trait. The causal gene will usually be found in the vicinity of the marker showing the highest association with the trait.

Finally, an association between a biallelic marker and a trait may occur when people with the trait and people without the trait correspond to genetically different subsets of the population who, coincidentally, also differ in the frequency of allele a (population stratification). This phenomenon may be avoided by using ethnically matched large heterogeneous samples.

Association studies are particularly suited to the efficient identification of genes that present common polymorphisms, and are involved in multifactorial traits whose frequency is relatively higher than that of diseases with monofactorial inheritance.

Association studies mainly consist of four steps: recruitment of trait-positive (T+) and control populations, preferably trait-negative (T−) populations with well-defined phenotypes, identification of a candidate region suspected of harboring a trait causing gene, identification of said gene among candidate genes in the region, and finally validation of mutation(s) responsible for the trait in said trait causing gene.

In a first step, the trait-positive should be well-defined, preferably the control phenotype is a well-defined trait-negative phenotype as well. In order to perform efficient and significant association studies such as those described herein, the trait under study should preferably follow a bimodal distribution in the population under study, presenting two clear non-overlapping phenotypes, trait-positive and trait-negative.

Nevertheless, in the absence of such a bimodal distribution (as may in fact be the case for complex genetic traits), any genetic trait may still be analyzed using the association method proposed herein by carefully selecting the individuals to be included in the trait-positive group and preferably the trait-negative phenotypic group as well. The selection procedure ideally involves selecting individuals at opposite ends of the non-bimodal phenotype spectrum of the trait under study, so as to include in these trait-positive and trait-negative populations individuals who clearly represent non-overlapping, preferably extreme phenotypes.

As discussed above, the definition of the inclusion criteria for the trait-positive and control populations is an important aspect of the present invention.

FIG. 3 shows, for a series of hypothetical sample sizes, the p-value significance obtained in association studies performed using individual markers from the high-density biallelic map, according to various hypotheses regarding the difference of allelic frequencies between the trait-positive and trait-negative samples. It indicates that, in all cases, samples ranging from 150 to 500 individuals are numerous enough to achieve statistical significance. It will be appreciated that bigger or smaller groups can be used to perform association studies according to the methods of the present invention.

In a second step, a marker/trait association study is performed that compares the genotype frequency of each biallelic marker in the above described trait-positive and trait-negative populations by means of a chi square statistical test (one degree of freedom). In addition to this single marker association analysis, a haplotype association analysis is performed to define the frequency and the type of the ancestral carrier haplotype. Haplotype analysis, by combining the informativeness of a set of biallelic markers increases the power of the association analysis, allowing false positive and/or negative data that may result from the single marker studies to be eliminated.

Genotyping can be performed using any method described in III, including the microsequencing procedure described in Example 8.

If a positive association with a trait is identified using an array of biallelic markers having a high enough density, the causal gene will be physically located in the vicinity of the associated markers, since the markers showing positive association with the trait are in linkage disequilibrium with the trait locus. Regions harboring a gene responsible for a particular trait which are identified through association studies using high density sets of biallelic markers will, on average, be 20-40 times shorter in length than those identified by linkage analysis.

Once a positive association is confirmed as described above, a third step consists of completely sequencing the BAC inserts harboring the markers identified in the association analyzes. These BACs are obtained through screening human genomic libraries with the markers probes and/or primers, as described above. Once a candidate region has been sequenced and analyzed, the functional sequences within the candidate region (e.g. exons, splice sites, promoters, and other potential regulatory regions) are scanned for mutations which are responsible for the trait by comparing the sequences of the functional regions in a selected number of trait-positive and trait-negative individuals using appropriate software. Tools for sequence analysis are further described in Example 9.

Finally, candidate mutations are then validated by screening a larger population of trait-positive and trait-negative individuals using genotyping techniques described below. Polymorphisms are confirmed as candidate mutations when the validation population shows association results compatible with those found between the mutation and the trait in the test population.

In practice, in order to define a region bearing a candidate gene, the trait-positive and trait-negative populations are genotyped using an appropriate number of biallelic markers. The markers may include one or more of the markers of SEQ ID Nos: 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto.

The markers used to define a region bearing a candidate gene may be distributed at an average density of 1 marker per 10-200 kb. Preferably, the markers used to define a region bearing a candidate gene are distributed at an average density of 1 marker every 15-150 kb. In further preferred embodiments, the markers used to define a region bearing a candidate gene are distributed at an average density of 1 marker every 20-100 kb. In yet another preferred embodiment, the markers used to define a region bearing a candidate gene are distributed at an average density of 1 marker every 100 to 150 kb. In a further highly preferred embodiment, the markers used to define a region bearing a candidate gene are distributed at an average density of 1 marker every 50 to 100 kb. In yet another embodiment, the biallelic markers used to define a region bearing a candidate gene are distributed at an average density of 1 marker every 25-50 kilobases. As mentioned above, in order to enhance the power of linkage disequilibrium based maps, in a preferred embodiment, the marker density of the map will be adapted to take the linkage disequilibrium distribution in the genomic region of interest into account.

In some embodiments, the initial identification of a candidate genomic region harboring a gene associated with a detectable phenotype may be conducted using a preliminary map containing a few thousand biallelic markers. Thereafter, the genomic region harboring the gene responsible for the detectable trait may be better delineated using a map containing a larger number of biallelic markers. Furthermore, the genomic region harboring the gene responsible for the detectable trait may be further delineated using a high density map of biallelic markers. Finally, the gene associated with the detectable trait may be identified and isolated using a very high density biallelic marker map.

Example 6 describes a procedure for identifying a candidate region harboring a gene associated with a detectable trait and provides simulated results for this procedure. It will be appreciated that although Example 6 compares the results of simulated analyzes using markers derived from maps having 3,000, 20,000, and 60,000 markers, the number of markers contained in the map is not restricted to these exemplary figures. Rather, Example 6 exemplifies the increasing refinement of the candidate region with increasing marker density. As increasing numbers of markers are used in the analysis, points in the association analysis become broad peaks. The gene associated with the detectable trait under investigation will lie within or near the region under the peak.

The statistical power of linkage disequilibrium mapping using a high density marker map is also reinforced by complementing the single point association analysis described above with a multi-marker association analysis of haplotype analysis described in IV. To improve the statistical power of the individual marker association analyses conducted as described above using maps of increasing marker densities, haplotype studies can be performed using groups of markers located in proximity to one another within regions of the genome. For example, using the methods described above in which the association of an individual marker with a detectable phenotype was analyzed using maps of 3,000 markers, 20,000 markers, and 60,000 markers, a series of haplotype studies can be performed using groups of contiguous markers from such maps or from maps having higher marker densities.

In a preferred embodiment, a series of successive haplotype studies including groups of markers spanning regions of more than 1 Mb may be performed. In some embodiments, the biallelic markers included in each of these groups may be located within a genomic region spanning less than 1 kb, from 1 to 5 kb, from 5 to 10 kb, from 10 to 25 kb, from 25 to 50 kb, from 50 to 150 kb, from 150 to 250 kb, from 250 to 500 kb, from 500 kb to 1 Mb, or more than 1 Mb. Preferably, the genomic regions containing the groups of biallelic markers used in the successive haplotype analyses are overlapping. It will be appreciated that the groups of biallelic markers need not completely cover the genomic regions of the above-specified lengths but may instead be obtained from incomplete contigs having one or more gaps therein. As discussed in further detail below, biallelic markers may be used in single point and haplotype association analyses regardless of the completeness of the corresponding physical contig harboring them.

Genome-wide mapping using association studies with dense enough arrays of markers permit a case-by-case best estimate of p-value significance thresholds. Given a test population comprising two ethnically matched trait-positive and control groups of about 50 to about 500 individuals or more, conducting the above described association studies will allow a p-value “cut-off” to be established by, for example, analyzing significant numbers of allele frequency differences or, in some cases where appropriate, running computer simulations or control studies as described in Examples 6, 15, and 26.

For a p-value above the threshold, a corresponding association between the trait and a studied marker will be deemed not significant, while for a p-value below such a threshold, said association will be deemed significant. If the p-value is significant, the genomic region around the marker will be further scrutinized for a trait-causing gene.

It is preferred that p-value significance thresholds be assessed for each case/control population comparison. Both the genetic distance between sampled population—“stratification”—and the dispersion due to random selection of samples may indeed influence the p-value significance thresholds.

It will be appreciated that the above approaches may be conducted on any scale (i.e. over the whole genome, a set of chromosomes, a single chromosome, a particular subchromosomal region, or any other desired portion of the genome). As mentioned above, once significance thresholds have been assessed, population sample sizes may be adapted as exemplified in FIG. 3.

Example 7 below illustrates the increase in statistical power brought to an association study by a haplotype analysis.

The results described in Examples 5 and 7, generated from individual and haplotype studies using a biallelic marker set of an average density equal to ca. 40 kb in the region of an Alzheimer's disease trait causing gene, indicate that all biallelic markers of sufficient informative content located within a ca. 200 kb genomic region around a trait-causing allele can potentially be successfully used to localize a trait causing gene with the methods provided by the present invention. This conclusion is further supported by the results obtained through measuring the linkage disequilibrium between markers 99-365-344 or 99-359-308 and ApoE 4 Site A marker within Alzheimer's patients: as one could predict since linkage disequilibrium is the supporting basis for association studies, linkage disequilibrium between these pairs of markers was enhanced in the diseased population vs. the control population. In a similar way as the haplotype analysis enhanced the significance of the corresponding association studies.

Once a given polymorphic site has been found and characterized as a biallelic marker according to the methods of the present invention, several methods can be used in order to determine the specific allele carried by an individual at the given polymorphic base as described in III.

Location of a Gene Associated with Detectable Traits

Once the candidate region has been delineated using the high density biallelic marker map, a sequence analysis process will allow the detection of all genes located within said region, together with a potential functional characterization of said genes. The identified functional features may allow preferred trait-causing candidates to be chosen from among the identified genes. More biallelic markers may then be generated within said candidate genes, and used to perform refined association studies that will support the identification of the trait causing gene. Sequence analysis processes are described in Example 9.

Examples 10-18 illustrate the application of the above methods using biallelic markers to identify a gene associated with a complex disease, prostate cancer, within a ca. 450 kb candidate region. Additional details of the identification of the gene associated with prostate cancer are provided in the U.S. patent application entitled “Prostate Cancer Gene” Ser. No. 08/996,306, the disclosure of which is incorporated herein by reference in its entirety.

The above methods were also used to identify biallelic markers in a gene which was an attractive candidate for a gene associated with asthma. Examples 19-26 show how the use of methods of the present invention allowed this gene to be identified as a gene responsible, at least partially, for asthma in the studied populations. Additional details of the identification of the gene associated with asthma are provided in U.S. Provisional Application Serial Nos. 60/081,893, the disclosure of which are incorporated herein by reference in its entirety

Alternatively, genes associated with detectable traits may be identified as follows. Candidate genomic regions suspected of harboring a gene associated with the trait may be identified using techniques such as those described herein. In such techniques, the allelic frequencies of biallelic markers are compared in nucleic acid samples derived from individuals expressing the detectable trait and individuals who do not express the detectable trait. In this manner, candidate genomic regions suspected of harboring a gene associated with the detectable trait under investigation are identified.

The existence of one or more genes associated with the detectable trait within the candidate region is confirmed by identifying more biallelic markers lying in the candidate region. A first haplotype analysis is performed for each possible combination of groups of biallelic markers within the genomic region suspected of harboring a trait-associated gene. For % example, each group may comprise three biallelic markers. For each of the groups of markers, the frequency of each possible haplotype (for groups of three markers there are 8 possible haplotypes) in individuals expressing the trait and individuals who do not express the trait is estimated. For example, the a haplotype estimation method is applied as described in IV. for example the haplotype frequencies may be estimated using the Expectation-Maximization method of Excoffier L and Slatkin M, Mol. Biol. Evol. 12:921-927 (1995), the disclosure of which is incorporated herein by reference in its entirety.

The frequencies of each of the possible haplotypes of the grouped markers (or each allele of individual markers) in individuals expressing the trait and individuals who do not express the trait are compared. For example, the frequencies may be compared by performing a chi-squared analysis. Within each group, the haplotype (or the allele of each individual marker) having the greatest association with the trait is selected. This process is repeated for each group of biallelic markers (or each allele of the individual markers) to generate a distribution of association values, which will be referred to herein as the “trait-associated” distribution.

A second haplotype analysis is performed for each possible combination of groups of biallelic markers within the genomic regions which are not suspected of harboring a trait-associated gene. For example, each group may comprise three biallelic markers. For each of the groups of markers, the frequency of each possible haplotype (for groups of three markers there are 8 possible haplotypes) in individuals expressing the trait and individuals who do not express the trait is estimated.

The frequencies of each of the possible haplotypes of the grouped markers (or each allele of individual markers) in individuals expressing the trait and individuals who do not express the trait are compared. For example, the frequencies may be compared by performing a chi-squared analysis. Within each group, the haplotype (or the allele of each individual marker) having the greatest association with the trait is selected. This process is repeated for each group of biallelic markers (or each allele of the individual markers) to generate a distribution of association values, which will be referred to herein as the “random” distribution.

The trait-associated distribution and the random distribution are then compared to one another to determine if there are significant differences between them. For example, the trait-associated distribution and the random distribution can be compared using either the Wilcoxon rank test (Noether, G. E. (1991) Introduction to statistics: “The nonparametric way”, Springer-Verlag, New York, Berlin, the disclosure of which is incorporated herein by reference in its entirety) or the Kolmogorov-Smirnov test (Saporta, G. (1990) “Probalites, analyse des donnees et statistiques” Technip editions, Paris, the disclosure of which is incorporated herein by reference in its entirety) or both the Wilcoxon rank test and the Kolmogorov-Smirnov test.

If the trait-associated distribution and the random distribution are found to be significantly different, the candidate genomic region is highly likely to contain a gene associated with the detectable trait. Accordingly, the candidate genomic region is evaluated more fully to isolate the trait-associated gene. Alternatively, if the trait-associated distribution and the random distribution are equal using the above analyses, the candidate genomic region is unlikely to contain a gene associated with the detectable trait. Accordingly, no further analysis of the candidate genomic region is performed.

While Examples 10 to 26 illustrate the use of the maps and markers of the present invention for identifying a new gene associated with a complex disease within a 2 Mb genomic region for establishing that a candidate gene is, at least partially, responsible for a disease, the maps and markers of the present invention may also be used to identify one or more biallelic markers or one or more genes associated with other detectable phenotypes, including drug response, drug toxicity, or drug efficacy. The biallelic markers used in such drug response analyses or shown, using the methods of the present invention to be associated with such traits, may lie within or near genes responsible for or partly responsible for a particular disease, for example a disease against which the drug is meant to act, or may lie within genomic regions which are not responsible for or partly responsible for a disease. In the context of the present invention, a “positive response” to a medicament can be defined as comprising a reduction of the symptoms related to the disease or condition to be treated. In the context of the present invention, a “negative response” to a medicament can be defined as comprising either a lack of positive response to the medicament which does not lead to a symptom reduction or to a side-effect observed following administration of the medicament.

Drug efficacy, response and tolerance/toxicity can be considered as multifactorial traits involving a genetic component in the same way as complex diseases such as Alzheimer's disease, prostate cancer, hypertension or diabetes. As such, the identification of genes involved in drug efficacy and toxicity could be achieved following a positional cloning approach, e.g. performing linkage analysis within families in order to obtain the subchromosomal location of the gene(s). However, this type of analysis is actually impractical in the case of drug responsiveness, due to the lack of availability of familial cases. In fact, the likelihood of having more than one individual in a particular family being exposed to the same drug at the same time is very low. Therefore, drug efficacy and toxicity can only be analyzed as sporadic traits.

In order to conduct association studies to analyze the individual response to a given drug in groups of patients affected with a disease, up to four groups are screened to determine their patterns of biallelic markers using the techniques described above. The four groups are:

Non-diseased or random controls,

Diseased patients/drug responders,

Diseased patients/drug non-responders, and

Diseased patients/drug side effects.

In preferred embodiments, the above mentioned groups are recruited according to phenotyping criteria having the characteristics described above, so that the phenotypes defining the different groups are non-overlapping, preferably extreme phenotypes. In highly preferred embodiments, such phenotyping criteria have the bimodal distribution described above.

The final number and composition of the groups for each drug association study is adapted to the distribution of the above described phenotypes within the studied population.

After selecting a suitable population, association and haplotype analyses may be performed as described herein to identify one or more biallelic markers associated with drug response, preferably drug toxicity or drug efficacy. The identification of such one or more biallelic markers allows one to conduct diagnostic tests to determine whether the administration of a drug to an individual will result in drug response, preferably drug toxicity, or drug efficacy.

The methods described above for identifying a gene associated with prostate cancer and biallelic markers indicative of a risk of suffering from asthma may be utilized to identify genes associated with other detectable phenotypes. In particular, the above methods may be used with any marker or combination of markers included in the maps of the present invention, including the biallelic markers of SEQ ID Nos.: 1 to 3809 or the sequences complementary thereto. As described above, the general strategy to perform the association studies using the maps and markers of the present invention is to scan two groups of individuals (trait-positive individuals and trait-negative controls) characterized by a well defined phenotype in order to measure the allele frequencies of the biallelic markers in each of these groups. Preferably, the frequencies of markers with inter-marker spacing of about 150 kb are determined in each group. More preferably, the frequencies of markers with inter-marker spacing of about 75 kb are determined in each group. Even more preferably, markers with inter-marker spacing of about 50 kb, about 37.5 kb, about 30 kb, or about 25 kb will be tested in each population.

In some embodiments, the frequencies of 1, 5, 10, 20, 50, 100, 500, 1000, 2000, 3000, or all of the biallelic markers of SEQ ID Nos.: 1 to 3908 or the sequences complementary thereto are measured in each population. In another embodiment, the frequencies of 1, 5, 10, 20, 50, 100, 500, 1000, 2000, or 3000 biallelic markers selected from the group consisting of biallelic markers which are in linkage disequilibrium with the biallelic markers of 1 to 3908 or the sequences complementary thereto are measured in each population. In some embodiments the frequencies of 1, 5, 10, 20, 50, 100, 500, 1000, 2000, or all of the biallelic markers of SEQ ID Nos.: 1 to 2260 or the sequences complementary thereto are measured in each population. In another embodiment, the frequencies of 1, 5, 10, 20, 50, 100, 500, 1000, or 2000 biallelic markers selected from the group consisting of biallelic markers which are in linkage disequilibrium with the biallelic markers of 1 to 2260 or the sequences complementary thereto are measured in each population. In some embodiments the frequencies of 1, 5, 10, 20, 50, 100, 500, 1000, or all of the biallelic markers of SEQ ID Nos.: 2261 to 3734 or the sequences complementary thereto are measured in each population. In another embodiment, the frequencies of 1, 5, 10, 20, 50, 100, 500, 1000 biallelic markers selected from the group consisting of biallelic markers which are in linkage disequilibrium with the biallelic markers of 2261 to 3734 or the sequences complementary thereto are measured in each population. In some embodiments the frequencies of 1, 5, 10, 20, 50, 100, or all of the biallelic markers of SEQ ID Nos.: 3735 to 3908 or the sequences complementary thereto are measured in each population. In another embodiment, the frequencies of 1, 5, 10, 20, 50, or 100 biallelic markers selected from the group consisting of biallelic markers which are in linkage disequilibrium with the biallelic markers of 3735 to 3908 or the sequences complementary thereto are measured in each population.

In some embodiments, the frequencies of about 20,000, or about 40,000 biallelic markers are determined in each population. In a highly preferred embodiment, the frequencies of about 60,000, about 80,000, about 100,000, or about 120,000 biallelic markers are determined in each population. In some embodiments, haplotype analyses may be run using groups of markers located within regions spanning less than 1 kb, from 1 to 5 kb, from 5 to 100 kb, from 10 to 25 kb, from 25 to 50 kb, from 50 to 150 kb, from 150 to 250 kb, from 250 to 500 kb, from 500 kb to 1 Mb, or more than 1 Mb.

Allele frequency can be measured using any genotyping method described herein including microsequencing techniques; preferred high throughput microsequencing procedures are further exemplified in III; it will be further appreciated that any other large scale genotyping method suitable with the intended purpose contemplated herein may also be used.

It will be appreciated that it is not necessary to use a full high density biallelic marker map in order to start a genome-wide association study. Maps having higher densities of biallelic markers (two or more markers per BAC, average inter-marker spacing of about 75 kb or less) may then be generated by starting first on those BACs for which a candidate association has been established at the first step.

In cases when one or more candidate regions have previously been delineated, such as cases where a particular gene or genomic region is suspected of being associated with a trait, local excerpts of biallelic marker maps having densities above one marker per 150 kb may be exploited using BACs harboring said genomic regions, or genes, or portions thereof. In these cases also, successive association studies may be performed using sets of biallelic markers showing increasing densities, preferably from about one every 150 kb to about one every 75 kb; more preferably, sets of markers with inter-marker spacing below about 50 kb, below about 37.5 kb, below about 30 kb, most preferably below about 25 kb, will be used.

Haplotype analyses may also be conducted using groups of biallelic markers within the candidate region. The biallelic markers included in each of these groups may be located within a genomic region spanning less than 1 kb, from 1 to 5 kb, from 5 to 10 kb, from 10 to 25 kb, from 25 to 50 kb, from 50 to 150 kb, from 150 to 250 kb, from 250 to 500 kb, from 500 kb to 1 Mb, or more than 1 Mb. It will be appreciated that the ordered DNA fragments containing these groups of biallelic markers need not completely cover the genomic regions of these lengths but may instead be incomplete contigs having one or more gaps therein. As discussed in further detail below, biallelic markers may be used in association studies and haplotype analyses regardless of the completeness of the corresponding physical contig harboring them, provided linkage disequilibrium between the markers can be assessed.

As described above, if a positive association with a trait, such as a disease, or a drug efficacy and/or toxicity, is identified using the biallelic markers and maps of the present invention, the maps will provide not only the confirmation of the association, but also a shortcut towards the identification of the gene involved in the trait under study. As described above, since the markers showing positive association to the trait are in linkage disequilibrium with the trait loci, the causal gene will be physically located in the vicinity of these markers. Regions identified through association studies using high density maps will on average have a 20-40 times shorter length than those identified by linkage analysis (2 to 20 Mb).

As described above, once a positive association is confirmed with the high density biallelic marker maps of the present invention, BACs from which the most highly associated markers were derived are completely sequenced and the mutations in the causal gene are searched by applying genomic analysis tools. As described above, once a region harboring a gene associated with a detectable trait has been sequenced and analyzed, the candidate functional regions (e.g. exons and splice sites, promoters and other regulatory regions) are scanned for mutations by comparing the sequences of a selected number of controls and cases, using adequate software.

In some embodiments, trait-positive samples being compared to identify causal mutations are selected among those carrying the ancestral haplotype; in these embodiments, control samples are chosen from individuals not carrying said ancestral haplotype.

In further embodiments, trait-positive samples being compared to identify causal mutations are selected among those showing haplotypes that are as close as possible to the ancestral haplotype; in these embodiments, control samples are chosen from individuals not carrying any of the haplotypes selected for the case population.

The maps and biallelic markers of the present invention may also be used to identify patterns of biallelic markers associated with detectable traits resulting from polygenic interactions. The analysis of genetic interaction between alleles at unlinked loci requires individual genotyping using the techniques described herein. The analysis of allelic interaction among a selected set of biallelic markers with appropriate p-values can be considered as a haplotype analysis, similar to those described in further details within the present invention.

IX. Use of Biallelic Markers to Identify Individuals Likely to Exhibit a Detectable Trait Associated with a Particular Allele of a Known Gene

In addition to their utility in searches for genes associated with detectable traits on a genome-wide, chromosome-wide, or subchromosomal level, the maps and biallelic markers of the present invention may be used in more targeted approaches for identifying individuals likely to exhibit a particular detectable trait or individuals who exhibit a particular detectable trait as a consequence of possessing a particular allele of a gene associated with the detectable trait. For example, the biallelic markers and maps of the present invention may be used to identify individuals who carry an allele of a known gene that is suspected of being associated with a particular detectable trait. In particular, the target genes may be genes having alleles which predispose an individual to suffer from a specific disease state. In other cases, the target genes may be genes having alleles that predispose an individual to exhibit a desired or undesired response to a drug or other pharmaceutical composition, a food, or any administered compound. The known gene may encode any of a variety of types of biomolecules. For example, the known genes targeted in such analyzes may be genes known to be involved in a particular step in a metabolic pathway in which disruptions may cause a detectable trait. Alternatively, the target genes may be genes encoding receptors or ligands which bind to receptors in which disruptions may cause a detectable trait, genes encoding transporters, genes encoding proteins with signaling activities, genes encoding proteins involved in the immune response, genes encoding proteins involved in hematopoesis, or genes encoding proteins involved in wound healing. It will be appreciated that the target genes are not limited to those specifically enumerated above, but may be any gene known to be or suspected of being associated with a detectable trait.

As previously mentioned, the maps and markers of the present invention may be used to identify genes associated with drug response. The biallelic markers of the present invention may also be used to select individuals for inclusion in the clinical trials of a drug. In some embodiments, the markers of SEQ ID Nos.: 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto may be used in targeted approaches to identify individuals at risk of developing a detectable trait, for example a complex disease or desired/undesired drug response, or to identify individuals exhibiting said trait. The present invention provides methods to establish putative associations between any of the biallelic markers described herein and any detectable traits, including those specifically described herein.

To use the maps and markers of the present invention in further targeted approaches, biallelic markers which are in linkage disequilibrium with any of the above disclosed markers may be identified. In cases where one or more biallelic markers of the present invention have been shown to be associated with a detectable trait, more biallelic markers in linkage disequilibrium with said associated biallelic markers may be generated and used to perform targeted approaches aiming at identifying individuals exhibiting, or likely to exhibit, said detectable trait, according to the methods provided herein.

Furthermore, in cases where a candidate gene is suspected of being associated with a particular detectable trait or suspected of causing the detectable trait, biallelic markers in linkage disequilibrium with said candidate gene may be identified and used in targeted approaches, such as the approaches utilized above for the asthma-associated gene and the Apo E gene.

Biallelic markers that are in linkage disequilibrium with markers associated with a detectable trait, or with genes associated with a detectable trait, or suspected of being so, are identified by performing single marker analyzes, haplotype association analyzes, or linkage disequilibrium measurements on samples from trait-positive and trait-negative individuals as described above using biallelic markers lying in the vicinity of the target marker or gene. In this manner, a single biallelic marker or a group of biallelic markers may be identified which indicate that an individual is likely to possess the detectable trait or does possess the detectable trait as a consequence of a particular allele of the target marker or gene.

Nucleic acid samples from individuals to be tested for predisposition to a detectable trait or possession of a detectable trait as a consequence of a particular allele of the target gene may be examined using the diagnostic methods described above.

Throughout this application, various publications, patents, and published patent applications are cited. The disclosures of the publications, patents, and published patent specifications referenced in this application are hereby incorporated by reference into the present disclosure in their entireties to more fully describe the state of the art to which this invention pertains.

EXAMPLES

Several of the methods of the present invention are described in the following examples, which are offered by way of illustration and not by way of limitation. Many other modifications and variations of the invention as herein set forth can be made without departing from the spirit and scope thereof and therefore only such limitations should be imposed as are indicated by the appended claims.

Example 1 Ordering of a BAC Library: Screening Clones with STSs

The BAC library is screened with a set of PCR-typeable STSs to identify clones containing the STSs. To facilitate PCR screening of several thousand clones, for example 200,000 clones, pools of clones are prepared.

Three-dimensional pools of the BAC libraries are prepared as described in Chumakov et al. and are screened for the ability to generate an amplification fragment in amplification reactions conducted using primers derived from the ordered STSs. (Chumakov et al. (1995), supra). A BAC library typically contains 200,000 BAC clones. Since the average size of each insert is 100-300 kb, the overall size of such a library is equivalent to the size of at least about 7 human genomes. This library is stored as an array of individual clones in 518 384-well plates. It can be divided into 74 primary pools (7 plates each). Each primary pool can then be divided into 48 subpools prepared by using a three-dimensional pooling system based on the plate, row and column address of each clone (more particularly, 7 subpools consisting of all clones residing in a given microtiter plate; 16 subpools consisting of all clones in a given row; 24 subpools consisting of all clones in a given column).

Amplification reactions are conducted on the pooled BAC clones using primers specific for the STSs. For example, the three dimensional pools may be screened with 45,000 STSs whose positions relative to one another and locations along the genome are known. Preferably, the three dimensional pools are screened with about 30,000 STSs whose positions relative to one another and locations along the genome are known. In a highly preferred embodiment, the three dimensional pools are screened with about 20,000 STSs whose positions relative to one another and locations along the genome are known.

Amplification products resulting from the amplification reactions are detected by conventional agarose gel electrophoresis combined with automatic image capturing and processing. PCR screening for a STS involves three steps: (1) identifying the positive primary pools; (2) for each positive primary pool, identifying the positive plate, row and column 'subpools′ to obtain the address of the positive clone; (3) directly confirming the PCR assay on the identified clone. PCR assays are performed with primers specifically defining the STS.

Screening is conducted as follows. First BAC DNA containing the genomic inserts is prepared as follows. Bacteria containing the BACs are grown overnight at 37° C. in 120 μl of LB containing chloramphenicol (12 μg/ml). DNA is extracted by the following protocol:

Centrifuge 10 min at 4° C. and 2000 rpm

Eliminate supernatant and resuspend pellet in 120 μl TE 10-2 (Tris HCl 10 mM, EDTA 2 mM)

Centrifuge 10 min at 4° C. and 2000 rpm

Eliminate supernatant and incubate pellet with 20 μl lyzozyme 1 mg/ml during 15 min at room temperature

Add 20 μl proteinase K 100 μg/ml and incubate 15 min at 60° C.

Add 8 μl DNAse 2U/μl and incubate 1 hr at room temperature

Add 100 μl TE 10-2 and keep at −80° C.

PCR assays are performed using the following protocol:

Final volume 15 μl BAC DNA 1.7 ng/μl MgCl₂ 2 mM dNTP (each) 200 μM primer (each) 2.9 ng/μl Ampli Taq Gold DNA polymerase 0.05 unit/μl PCR buffer (10× = 0.1 M TrisHCl pH 8.3 0.5 M KCl 1×

The amplification is performed on a Genius II thermocycler. After heating at 95° C. for 10 min, 40 cycles are performed. Each cycle comprises: 30 sec at 95° C., 54° C. for 1 min, and 30 sec at 72° C. For final elongation, 10 min at 72° C. end the amplification. PCR products are analyzed on 1% agarose gel with 0.1 mg/ml ethidium bromide.

Alternatively, a YAC (Yeast Artificial Chromosome) library can be used. The very large insert size, of the order of 1 megabase, is the main advantage of the YAC libraries. The library can typically include about 33,000 YAC clones as described in Chumakov et al. (1995, supra). The YAC screening protocol may be the same as the one used for BAC screening.

The known order of the STSs is then used to align the BAC inserts in an ordered array (contig) spanning the whole human genome. If necessary new STSs to be tested can be generated by sequencing the ends of selected BAC inserts. Subchromosomal localization of the BACs can be established and/or verified by fluorescence in situ hybridization (FISH), performed on metaphasic chromosomes as described by Cherif et al. 1990 and in Example 3 below. BAC insert size may be determined by Pulsed Field Gel Electrophoresis after digestion with the restriction enzyme NotI.

Finally, a minimally overlapping set of BAC clones, with known insert size and subchromosomal location, covering the entire genome, a set of chromosomes, a single chromosome, a particular subchromosomal region, or any other desired portion of the genome is selected from the DNA library. For example, the BAC clones may cover at least 100 kb of contiguous genomic DNA, at least 250 kb of contiguous genomic DNA, at least 500 kb of contiguous genomic DNA, at least 2 Mb of contiguous genomic DNA, at least 5 Mb of contiguous genomic DNA, at least 10 Mb of contiguous genomic DNA, or at least 20 Mb of contiguous genomic DNA.

Example 2 Screening BAC Libraries with Biallelic Markers

Amplification primers enabling the specific amplification of DNA fragments carrying the biallelic markers, including the map-related biallelic markers of the invention, may be used to screen clones in any genomic DNA library, preferably the BAC libraries described above for the presence of the biallelic markers.

Pairs of primers of SEQ ID Nos: 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 were designed which allow the amplification of fragments carrying the biallelic markers of SEQ ID Nos: 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto. The amplification primers of SEQ ID Nos: 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773 may be used to screen clones in a genomic DNA library for the presence of the biallelic markers of SEQ ID Nos: 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto.

It will be appreciated that amplification primers for the biallelic markers of SEQ ID Nos: 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 need not be identical to the primers of SEQ ID Nos: 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773. Rather, they can be any other primers allowing the specific amplification of any DNA fragment carrying the markers and may be designed using techniques familiar to those skilled in the art. The amplification primers may be oligonucleotides of 8, 10, 15, 20 or more bases in length which enable the amplification of any fragment carrying the polymorphic site in the markers. The polymorphic base may be in the center of the amplification product or, alternatively, it may be located off-center. For example, in some embodiments, the amplification product produced using these primers may be at least 100 bases in length (i.e. 50 nucleotides on each side of the polymorphic base in amplification products in which the polymorphic base is centrally located). In other embodiments, the amplification product produced using these primers may be at least 500 bases in length (i.e. 250 nucleotides on each side of the polymorphic base in amplification products in which the polymorphic base is centrally located). In still further embodiments, the amplification product produced using these primers may be at least 1000 bases in length (i.e. 500 nucleotides on each side of the polymorphic base in amplification products in which the polymorphic base is centrally located). Amplification primers such as those described above are included within the scope of the present invention.

The localization of biallelic markers on BAC clones is performed essentially as described in Example 1.

The BAC clones to be screened are distributed in three dimensional pools as described in Example 1.

Amplification reactions are conducted on the pooled BAC clones using primers specific for the biallelic markers to identify BAC clones which contain the biallelic markers, using procedures essentially similar to those described in Example 1.

Amplification products resulting from the amplification reactions are detected by conventional agarose gel electrophoresis combined with automatic image capturing and processing. PCR screening for a biallelic marker involves three steps: (1) identifying the positive primary pools; (2) for each positive primary pools, identifying the positive plate, row and column ‘subpools’ to obtain the address of the positive clone; (3) directly confirming the PCR assay on the identified clone. PCR assays are performed with primers defining the biallelic marker.

Screening is conducted as follows. First BAC DNA is isolated as follows. Bacteria containing the genomic inserts are grown overnight at 37° C. in 120 μl of LB containing chloramphenicol (12 μg/ml). DNA is extracted by the following protocol:

Centrifuge 10 min at 4° C. and 2000 rpm

Eliminate supernatant and resuspend pellet in 120 μl TE 10-2 (Tris HCl 10 mM, EDTA 2 mM)

Centrifuge 10 min at 4° C. and 2000 rpm

Eliminate supernatant and incubate pellet with 20 μl lyzozyme 1 mg/ml during 15 min at room temperature

Add 20 μl proteinase K 100 μg/ml and incubate 15 min at 60° C.

Add 8 μl DNAse 2U/μl and incubate 1 hr at room temperature

Add 100 μl TE 10-2 and keep at −80° C.

PCR assays are performed using the following protocol:

Final volume 15 μl BAC DNA 1.7 ng/μl MgCl₂ 2 mM dNTP (each) 200 μM primer (each) 2.9 ng/μl Ampli Taq Gold DNA polymerase 0.05 unit/μl PCR buffer (10× = 0.1 M TrisHCl pH 8.3 0.5 M KCl 1×

The amplification is performed on a Genius II thermocycler. After heating at 95° C. for 10 min, 40 cycles are performed. Each cycle comprises: 30 sec at 95° C., 54° C. for 1 min, and 30 sec at 72° C. For final elongation, 10 min at 72° C. end the amplification. PCR products are analyzed on 1% agarose gel with 0.1 mg/ml ethidium bromide.

Example 3 Assignment of Biallelic Markers to Subchromosomal Regions

Metaphase chromosomes are prepared from phytohemagglutinin (PHA)-stimulated blood cell donors. PHA-stimulated lymphocytes from healthy males are cultured for 72 h in RPMI-1640 medium. For synchronization, methotrexate (10 mM) is added for 17 h, followed by addition of 5-bromodeoxyuridine (5-BudR, 0.1 mM) for 6 h. Colcemid (1 mg/ml) is added for the last 15 min before harvesting the cells. Cells are collected, washed in RPMI, incubated with a hypotonic solution of KCl (75 mM) at 37° C. for 15 min and fixed in three changes of methanol:acetic acid (3:1). The cell suspension is dropped onto a glass slide and air-dried.

BAC clones carrying the biallelic markers used to construct the maps of the present invention (including the biallelic markers of SEQ ID Nos: 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto) can be isolated as described above. These BACs or portions thereof, including fragments carrying said biallelic markers, obtained for example from amplification reactions using pairs of primers of SEQ ID Nos: 3935 to 7842, 3935 to 6194, 6195 to 7668, 7669 to 7842, 7866 to 11773, 7866 to 10125, 10126 to 11599, and 11600 to 11773, can be used as probes to be hybridized with metaphasic chromosomes. It will be appreciated that the hybridization probes to be used in the contemplated method may be generated using alternative methods well known to those skilled in the art. Hybridization probes may have any length suitable for this intended purpose.

Probes are then labeled with biotin-16 dUTP by nick translation according to the manufacturer's instructions (Bethesda Research Laboratories, Bethesda, Md.), purified using a Sephadex G-50 column (Pharmacia, Upssala, Sweden) and precipitated. Just prior to hybridization, the DNA pellet is dissolved in hybridization buffer (50% formamide, 2×SSC, 10% dextran sulfate, 1 mg/ml sonicated salmon sperm DNA, pH 7) and the probe is denatured at 70° C. for 5-10 min.

Slides kept at −20° C. are treated for 1 h at 37° C. with RNase A (100 mg/ml), rinsed three times in 2×SSC and dehydrated in an ethanol series. Chromosome preparations are denatured in 70% formamide, 2×SSC for 2 min at 70° C., then dehydrated at 4° C. The slides are treated with proteinase K (10 mg/100 ml in 20 mM Tris-HCl, 2 mM CaCl₂) at 37° C. for 8 min and dehydrated. The hybridization mixture containing the probe is placed on the slide, covered with a coverslip, sealed with rubber cement and incubated overnight in a humid chamber at 37° C. After hybridization and post-hybridization washes, the biotinylated probe is detected by avidin-FITC and amplified with additional layers of biotinylated goat anti-avidin and avidin-FITC. For chromosomal localization, fluorescent R-bands are obtained as previously described (Cherif et al., (1990) supra.). The slides are observed under a LEICA fluorescence microscope (DMRXA). Chromosomes are counterstained with propidium iodide and the fluorescent signal of the probe appears as two symmetrical yellow-green spots on both chromatids of the fluorescent R-band chromosome (red). Thus, a particular biallelic marker may be localized to a particular cytogenetic R-band on a given chromosome.

The above procedure was used to confirm the subchromosomal location of many of the BAC clones harboring the markers obtained above. In particular, several of the markers were assigned to subchromosomal regions of chromosome 21. Simple identification numbers were attributed to each BAC from which the markers are derived. FIG. 1 is a cytogenetic map of chromosome 21 indicating the subchromosomal regions therein. Amplification primers for generating amplification products containing the polymorphic bases of these markers are also provided in the accompanying sequence listing. In addition, microsequencing primers for use in determining the identities of the polymorphic bases of these biallelic markers are provided in the accompanying Sequence Listing.

The rate at which biallelic markers may be assigned to subchromosomal regions may be enhanced through automation. For example, probe preparation may be performed in a microtiter plate format, using adequate robots. The rate at which biallelic markers may be assigned to subchromosomal regions may be enhanced using techniques which permit the in situ hybridization of multiple probes on a single microscope slide, such as those disclosed in Larin et al., Nucleic Acids Research 22: 3689-3692 (1994), the disclosure of which is incorporated herein by reference in its entirety. In the largest test format described, different probes were hybridized simultaneously by applying them directly from a 96-well microtiter dish which was inverted on a glass plate. Software for image data acquisition and analysis that is adapted to each optical system, test format, and fluorescent probe used, can be derived from the system described in Lichter et al. Science 247: 64-69 (1990), the disclosure of which is incorporated herein by reference in its entirety. Such software measures the relative distance between the center of the fluorescent spot corresponding to the hybridized probe and the telomeric end of the short arm of the corresponding chromosome, as compared to the total length of the chromosome. The rate at which biallelic markers are assigned to subchromosomal locations may be further enhanced by simultaneously applying probes labeled with different flouorescent tags to each well of the 96 well dish. A further benefit of conducting the analysis on one slide is that it facilitates automation, since a microscope having a moving stage and the capability of detecting fluorescent signals in different metaphase chromosomes could provide the coordinates of each probe on the metaphase chromosomes distributed on the 96 well dish.

Example 4 below describes an alternative method to position biallelic markers which allows their assignment to human chromosomes.

Example 4 Assignment of Biallelic Markers to Human Chromosomes

The biallelic markers used to construct the maps of the present invention, including the biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto, may be assigned to a human chromosome using monosomal analysis as described below.

The chromosomal localization of a biallelic marker can be performed through the use of somatic cell hybrid panels. For example 24 panels, each panel containing a different human chromosome, may be used (Russell et al., Somat Cell Mol. Genet 22:425-431 (1996); Drwinga et al., Genomics 16:311-314 (1993), the disclosures of which are incorporated herein by reference in their entireties).

The biallelic markers are localized as follows. The DNA of each somatic cell hybrid is extracted and purified. Genomic DNA samples from a somatic cell hybrid panel are prepared as follows. Cells are lysed overnight at 42° C. with 3.7 ml of lysis solution composed of:

3 ml TE 10-2 (Tris HCl 10 mM, EDTA 2 mM)/NaCl 0.4 M

200 μl SDS 10%

500 μl K-proteinase (2 mg K-proteinase in TE 10-2/NaCl 0.4 M)

For the extraction of proteins, 1 ml saturated NaCl (6M) (1/3.5 v/v) is added. After vigorous agitation, the solution is centrifuged for 20 min at 10,000 rpm. For the precipitation of DNA, 2 to 3 volumes of 100% ethanol are added to the previous supernatant, and the solution is centrifuged for 30 min at 2,000 rpm. The DNA solution is rinsed three times with 70% ethanol to eliminate salts, and centrifuged for 20 min at 2,000 rpm. The pellet is dried at 37° C., and resuspended in 1 ml TE 10-1 or 1 ml water. The DNA concentration is evaluated by measuring the OD at 260 nm (1 unit OD=50 μg/ml DNA). To determine the presence of proteins in the DNA solution, the OD₂₆₀/OD₂₈₀ ratio is determined. Only DNA preparations having a OD₂₆₀/OD₂₈₀ ratio between 1.8 and 2 are used in the PCR assay.

Then, a PCR assay is performed on genomic DNA with primers defining the biallelic marker. The PCR assay is performed as described above for BAC screening. The PCR products are analyzed on a 1% agarose gel containing 0.2 mg/ml ethidium bromide.

Example 5 Measurement of Linkage Disequilibrium

As originally reported by Strittmater et al. and by Saunders et al. in 1993, the Apo E e4 allele is strongly associated with both late-onset familial and sporadic Alzheimer's disease. (Saunders, A. M. Lancet 342: 710-711 (1993) and Strittmater, W. J. et al., Proc. Natl. Acad. Sci. U.S.A. 90: 1977-1981 (1993), the disclosures of which are incorporated herein by reference in their entireties). The 3 major isoforms of human Apolipoprotein E (apoE2, -E3, and -E4), as identified by isoelectric focusing, are coded for by 3 alleles (e 2, 3, and 4). The e 2, e 3, and e 4 isoforms differ in amino acid sequence at 2 sites, residue 112 (called site A) and residue 158 (called site B). The ancestral isoform of the protein is Apo E3, which at sites A/B contains cysteine/arginine, while ApoE2 and -E4 contain cysteine/cysteine and arginine/arginine, respectively (Weisgraber, K. H. et al., J. Biol. Chem. 256: 9077-9083 (1981); Rall, S. C. et al., Proc. Natl. Acad. Sci. U.S.A. 79: 4696-4700 (1982), the disclosures of which are incorporated herein by reference in their entireties).

Apo E e 4 is currently considered as a major susceptibility risk factor for Alzheimer's disease development in individuals of different ethnic groups (specially in Caucasians and Japanese compared to Hispanics or African Americans), across all ages between 40 and 90 years, and in both men and women, as reported recently in a study performed on 5930 Alzheimer's disease patients and 8607 controls (Farrer et al., JAMA 278:1349-1356 (1997), the disclosure of which is incorporated herein by reference in its entirety). More specifically, the frequency of a C base coding for arginine 112 at site A is significantly increased in Alzheimer's disease patients.

Although the mechanistic link between Apo E e 4 and neuronal degeneration characteristic of Alzheimer's disease remains to be established, current hypotheses suggest that the Apo E genotype may influence neuronal vulnerability by increasing the deposition and/or aggregation of the amyloid beta peptide in the brain or by indirectly reducing energy availability to neurons by promoting atherosclerosis.

Using the methods of the present invention, biallelic markers that are in the vicinity of the Apo E site A were generated and the association of one of their alleles with Alzheimer's disease was analyzed. An Apo E public marker (stSG94) was used to screen a human genome BAC library as previously described. A BAC, which gave a unique FISH hybridization signal on chromosomal region 19q13.2.3, the chromosomal region harboring the Apo E gene, was selected for finding biallelic markers in linkage disequilibrium with the Apo E gene as follows.

This BAC contained an insert of 205 kb that was subcloned as previously described. Fifty BAC subclones were randomly selected and sequenced. Twenty five subclone sequences were selected and used to design twenty five pairs of PCR primers allowing 500 bp-amplicons to be generated. These PCR primers were then used to amplify the corresponding genomic sequences in a pool of DNA from 100 unrelated individuals (blood donors of French origin) as already described.

Amplification products from pooled DNA were sequenced and analyzed for the presence of biallelic polymorphisms, as already described. Five amplicons were shown to contain a polymorphic base in the pool of 100 unrelated individuals, and therefore these polymorphisms were selected as random biallelic markers in the vicinity of the Apo E gene. The sequences of both alleles of these biallelic markers (99-344-439; 99-366-274, 99-359-308; 99-355-219; 99-365-344;) correspond to SEQ ID Nos: 3909 to 3913. Corresponding pairs of amplification primers for generating amplicons containing these biallelic markers can be chosen from those listed as SEQ ID Nos: 7843 to 7847 and 11774 to 11778.

An additional pair of primers (SEQ ID Nos: 3124 and 4169) was designed that allows amplification of the genomic fragment carrying the biallelic polymorphism corresponding to the ApoE marker (99-2452-54; C/T; designated SEQ ID NO: 3914 in the accompanying Sequence Listing; publicly known as Apo E site A (Weisgraber et al. (1981), supra; Rall et al. (1982), supra) to be amplified.

The five random biallelic markers plus the Apo E site A marker were physically ordered by PCR screening of the corresponding amplicons using all available BACs originally selected from the genomic DNA libraries, as previously described, using the public Apo E marker stSG94. The amplicon's order derived from this BAC screening is as follows: (99-344-439/99-366-274)-(99-365-344/99-2452-54)-99-359-308-99-355-219, where parentheses indicate that the exact order of the respective amplicons couldn't be established.

Linkage disequilibrium among the six biallelic markers (five random markers plus the Apo E site A) was determined by genotyping the same 100 unrelated individuals from whom the random biallelic markers were identified.

DNA samples and amplification products from genomic PCR were obtained in similar conditions as those described above for the generation of biallelic markers, and subjected to automated microsequencing reactions using fluorescent ddNTPs (specific fluorescence for each ddNTP) and the appropriate microsequencing primers having a 3′ end immediately upstream of the polymorphic base in the biallelic markers. Once specifically extended at the 3′ end by a DNA polymerase using the complementary fluorescent dideoxynucleotide analog (thermal cycling), the microsequencing primer was precipitated to remove the unincorporated fluorescent ddNTPs. The reaction products were analyzed by electrophoresis on ABI 377 sequencing machines. Results were automatically analyzed by an appropriate software further described in Example 8.

Linkage disequilibrium (LD) between all pairs of biallelic markers (M_(i), M_(j)) was calculated for every allele combination (Mi1,Mj1; Mi1,Mj2; Mi2,Mj1; Mi2,Mj2) according to the maximum likelihood estimate (MLE) for delta (the composite linkage disequilibrium coefficient). The results of the linkage disequilibrium analysis between the Apo E Site A marker and the five new biallelic markers (99-344-439; 99-355-219; 99-359-308; 99-365-344; 99-366-274) are summarized in Table 2 below:

TABLE 2 d × 100 SEQ ID Nos of APOE Site A SEQ ID Nos of the the amplification Markers 99-2452-54 biallelic Markers Primers ApoE Site A 3914 7848; 11779 99-2452-54 99-344-439 1 3909 7843, 11774 99-366-274 1 3910 7844, 11775 99-365-344 8 3913 7847, 11778 99-359-308 2 3911 7845, 11776 99-355-219 1 3912 7846, 11777

The above linkage disequilibrium results indicate that among the five biallelic markers randomly selected in a region of about 200 kb containing the Apo E gene, marker 99-365-344T is in relatively strong linkage disequilibrium with the Apo E site A allele (99-2452-54C).

Therefore, since the Apo E site A allele is associated with Alzheimer's disease, one can predict that the T allele of marker 99-365-344 will probably be found associated with Alzheimer's disease. In order to test this hypothesis, the biallelic markers of SEQ ID Nos: 3909 to 3913 were used in association studies as described below.

225 Alzheimer's disease patients were recruited according to clinical inclusion criteria based on the MMSE test. The 248 control cases included in this study were both ethnically- and age-matched to the affected cases. Both affected and control individuals corresponded to unrelated cases. The identities of the polymorphic bases of each of the biallelic markers was determined in each of these individuals using the methods described above. Techniques for conducting association studies are further described below.

The results of this study are summarized in Table 3 below:

TABLE 3 ASSOCIATION DATA Difference in allele frequency between individuals with Alzheimer's and MARKER control individuals Corresponding p-value 99-344-439 3.3% 9.54 E-02 99-366-274 1.6% 2.09 E-01 99-365-344 17.7%  6.9 E-10 99-2452-54 (ApoE Site A) 23.8% 3.95 E-21 99-359-308 0.4%  9.2 E-01 99-355-219 2.5% 2.54 E-01

The frequency of the Apo E site A allele in both Alzheimer's disease cases and controls was found in agreement with that previously reported (ca. 10% in controls and ca. 34% in Alzheimer's disease cases, leading to a 24% difference in allele frequency), thus validating the Apo E e4 association in the populations used for this study.

Moreover, as predicted from the linkage disequilibrium analysis (Table 3), a significant association of the T allele of marker 99-365/344 with Alzheimer's disease cases (18% increase in the T allele frequency in Alzheimer's disease cases compared to controls, p value for this difference=6.9 E-10) was observed.

The above results indicate that any marker in linkage disequilibrium with one given marker associated with a trait will be associated with the trait. It will be appreciated that, though in this case the ApoE Site A marker is the trait-causing allele (TCA) itself, the same conclusion could be drawn with any other non trait-causing allele marker associated with the studied trait.

These results further indicate that conducting association studies with a set of biallelic markers randomly generated within a candidate region at a sufficient density (here about one biallelic marker every 40 kb on average), allows the identification of at least one marker associated with the trait.

In addition, these results correlate with the physical order of the six biallelic markers contemplated within the present example (see above): marker 99-365/344, which had been found to be the closest in terms of physical distance to the ApoE Site A marker, also shows the strongest linkage disequilibrium with the Apo E site A marker.

In order to further refine the relationship between physical distance and linkage disequilibrium between biallelic markers, a ca. 450 kb fragment from a genomic region on chromosome 8 was fully sequenced.

LD within ca. 230 pairs of biallelic markers derived therefrom was measured in a random French population and analyzed as a function of the known physical inter-marker spacing. This analysis confirmed that, on average, linkage disequilibrium between 2 biallelic markers correlates with the physical distance that separates them. It further indicated that linkage disequilibrium between 2 biallelic markers tends to decrease when their spacing increases. More particularly, linkage disequilibrium between 2 biallelic markers tends to decrease when their inter-marker distance is greater than 50 kb, and is further decreased when the inter-marker distance is greater than 75 kb. It was further observed that when 2 biallelic markers were further than 150 kb apart, most often no significant linkage disequilibrium between them could be evidenced. It will be appreciated that the size and history of the sample population used to measure linkage disequilibrium between markers may influence the distance beyond which linkage disequilibrium tends not to be detectable. Assuming that linkage disequilibrium can be measured between markers spanning regions up to an average of 150 kb long, biallelic marker maps will allow genome-wide linkage disequilibrium mapping, provided they have an average inter-marker distance lower than 150 kb.

Example 6 Identification of a Candidate Region Harboring a Gene Associated with a Detectable Trait

The initial identification of a candidate genomic region harboring a gene associated with a detectable trait may be conducted using a genome-wide map comprising about 20,000 biallelic markers. The candidate genomic region may be further defined using a map having a higher marker density, such as a map comprising about 40,000 markers, about 60,000 markers, about 80,000 markers, about 100,000 markers, or about 120,000 markers.

The use of high density maps such as those described above allows the identification of genes which are truly associated with detectable traits, since the coincidental associations will be randomly distributed along the genome while the true associations will map within one or more discrete genomic regions. Accordingly, biallelic markers located in the vicinity of a gene associated with a detectable trait will give rise to broad peaks in graphs plotting the frequencies of the biallelic markers in trait-positive individuals versus control individuals. In contrast, biallelic markers which are not in the vicinity of the gene associated with the detectable trait will produce unique points in such a plot. By determining the association of several markers within the region containing the gene associated with the detectable trait, the gene associated with the detectable trait can be identified using an association curve which reflects the difference between the allele frequencies within the trait-positive and control populations for each studied marker. The gene associated with the detectable trait will be found in the vicinity of the marker showing the highest association with the trait.

FIGS. 4, 5, and 6 provide a simulated illustration of the above principles. As illustrated in FIG. 4, an association analysis conducted with a map comprising about 3,000 biallelic markers yields a group of points. However, when an association analysis is performed using a denser map which includes additional biallelic markers, the points become broad peaks indicative of the location of a gene associated with a detectable trait. For example, the biallelic markers used in the initial association analysis may be obtained from a map comprising about 20,000 biallelic markers, as illustrated by the simulation results shown in FIG. 5. In some embodiments, one or more of the biallelic markers of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto are used in the association analysis.

In the simulated results of FIG. 4, the association analysis with 3,000 markers suggests peaks near markers 9 and 17.

Next, a second analysis is performed using additional markers in the vicinity of markers 9 and 17, as illustrated in the simulated results of FIG. 5, using a map of about 20,000 markers. This step again indicates an association in the close vicinity of marker 17, since more markers in this region show an association with the trait. However, none of the additional markers around marker 9 shows a significant association with the trait, which makes marker 9 a potential false positive. In some embodiments, one or more of the biallelic markers selected from the group consisting of SEQ ID Nos. 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto are used in the second analysis. In order to further test the validity of these two suspected associations, a third analysis may be obtained with a map comprising about 60,000 biallelic markers. In some embodiments, one or more of the biallelic markers selected from the group consisting of SEQ ID Nos: 1 to 3908, 1 to 2260, 2261 to 3374, 3735 to 3908 or the sequences complementary thereto are used in the third association analysis. In the simulated results of FIG. 6, more markers lying around marker 17 exhibit a high degree of association with the detectable trait. Conversely, no association is confirmed in the vicinity of marker 9. The genomic region surrounding marker 17 can thus be considered a candidate region for the potential trait of this simulation.

Example 7 Haplotype Analysis: Identification of Biallelic Markers Delineating a Genomic Region Associated with Alzheimer's Disease (AD)

As shown in Table 3 within Example 5, at an average map density of one marker per 40 kb only one marker (99-365-344) out of five random biallelic markers from a ca. 200 kb genomic region around the Apo E gene showed a clear association to Alzheimer's disease (delta allelic frequency in cases and controls=18%; p value=6.9 E-10). The allelic frequencies of the other four random markers were not significantly different between Alzheimer's disease cases and controls (p-values≧E-01). However, since linkage disequilibrium can usually be detected between markers located further apart than an average 40 kb as previously discussed, one should expect that, performing an association study with a local excerpt of a biallelic marker map covering ca. 200 kb with an average inter-marker distance of ca. 40 kb should allow the identification of more than one biallelic marker associated with Alzheimer's disease.

A haplotype analysis was thus performed using the biallelic markers 99-344-439; 99-355-219; 99-359-308; 99-365-344; and 99-366-274 (of SEQ ID Nos: 3909 to 3919).

In a first step, marker 99-365-344 that was already found associated with Alzheimer's disease was not included in the haplotype study. Only biallelic markers 99-344-439, 99-355-219, 99-359-308, and 99-366-274, which did not show any significant association with Alzheimer's disease when taken individually, were used. This first haplotype analysis measured frequencies of all possible two-, three-, or four-marker haplotypes in the Alzheimer's disease case and control populations. As shown in FIG. 7, there was one haplotype among all the potential different haplotypes based on the four individually non-significant markers (“haplotype 8”, TAGG comprising SEQ ID No. 3910 with the T allele of marker 99-366-274, SEQ ID No. 3909 with the A allele of marker 99-344-439, SEQ ID No. 3911 with the G allele of marker 99-359-308 and SEQ ID No. 3912 which is the G allele of marker 99-355-219), that was present at statistically significant different frequencies in the Alzheimer's disease case and control populations (D=12%; p value=2.05 E-06). Moreover, a significant difference was already observed for a three-marker haplotype included in the above mentioned “haplotype 8” (“haplotype 7”, TGG, D=10%; p value=4.76 E-05). Haplotype 7 comprises SEQ ID No. 3910 with the T allele of marker 99-366-274, SEQ ID No. 3911 with the G allele of marker 99-359-308 and SEQ ID No. 3912 with the G allele of marker 99-355-219). The haplotype association analysis thus clearly increased the statistical power of the individual marker association studies by more than four orders of magnitude when compared to single-marker analysis from p values≧E-01 for the individual markers to p value<2 E-06 for the four-marker “haplotype 8”. See Table 3.

The significance of the values obtained for this haplotype association analysis was evaluated by the following computer simulation. The genotype data from the Alzheimer's disease cases and the unaffected controls were pooled and randomly allocated to two groups which contained the same number of individuals as the case/control groups used to produce the data summarized in FIG. 7. A four-marker haplotype analysis (99-344-439; 99-355-219; 99-359-308; and 99-366-274) was run on these artificial groups. This experiment was reiterated 100 times and the results are shown in FIG. 8. No haplotype among those generated was found for which the p-value of the frequency difference between both populations was more significant than 1 E-05. In addition, only 4% of the generated haplotypes showed p-values lower than 1 E-04. Since both these p-value thresholds are less significant than the 2 E-06 p-value showed by “haplotype 8”, this haplotype can be considered significantly associated with Alzheimer's disease.

In a second step, marker 99-365-344 was included in the haplotype analyzes. The frequency differences between the affected and non affected populations was calculated for all two-, three-, four- or five-marker haplotypes involving markers: 99-344-439; 99-355-219; 99-359-308; 99-366-274; and 99-365-344. The most significant p-values obtained in each category of haplotype (involving two, three, four or five markers) were examined depending on which markers were involved or not within the haplotype. This showed that all haplotypes which included marker 99-36S-344 showed a significant association with Alzheimer's disease (p-values in the range of E-04 to E-11).

An additional way of evaluating the significance of the values obtained in the haplotype association analysis was to perform a similar Alzheimer's disease case-control study on biallelic markers generated from BACs containing inserts corresponding to genomic regions derived from chromosomes 13 or 21 and not known to be involved in Alzheimer's disease. Performing similar haplotype and individual association analyzes as those described above and in Example 10 did not generate any significant association results (all p-values for haplotype analyzes were less significant than E-03; all p-values for single marker association studies were less significant than E-02).

Example 8 Genotyping of Biallelic Markers Using Microsequencing Procedures

Several microsequencing protocols conducted in liquid phase are well known to those skilled in the art. A first possible detection analysis allowing the allele characterization of the microsequencing reaction products relies on detecting fluorescent ddNTP-extended microsequencing primers after gel electrophoresis. A first alternative to this approach consists in performing a liquid phase microsequencing reaction, the analysis of which may be carried out in solid phase.

For example, the microsequencing reaction may be performed using 5′-biotinylated oligonucleotide primers and fluorescein-dideoxynucleotides. The biotinylated oligonucleotide is annealed to the target nucleic acid sequence immediately adjacent to the polymorphic nucleotide position of interest. It is then specifically extended at its 3′-end following a PCR cycle, wherein the labeled dideoxynucleotide analog complementary to the polymorphic base is incorporated. The biotinylated primer is then captured on a microtiter plate coated with streptavidin. The analysis is thus entirely carried out in a microtiter plate format. The incorporated ddNTP is detected by a fluorescein antibody—alkaline phosphatase conjugate.

In practice this microsequencing analysis is performed as follows. 20 μl of the microsequencing reaction is added to 80 μl of capture buffer (SSC 2×, 2.5% PEG 8000, 0.25 M Tris pH7.5, 1.8% BSA, 0.05% Tween 20) and incubated for 20 minutes on a microtiter plate coated with streptavidin (Boehringer). The plate is rinsed once with washing buffer (0.1 M Tris pH 7.5, 0.1 M NaCl, 0.1% Tween 20). 100 μl of anti-fluorescein antibody conjugated with phosphatase alkaline, diluted 1/5000 in washing buffer containing 1.8% BSA is added to the microtiter plate. The antibody is incubated on the microtiter plate for 20 minutes. After washing the microtiter plate four times, 100 μl of 4-methylumbelliferyl phosphate (Sigma) diluted to 0.4 mg/ml in 0.1 M diethanolamine pH 9.6, 10mM MgCl₂ are added. The detection of the microsequencing reaction is carried out on a fluorimeter (Dynatech) after 20 minutes of incubation.

As another alternative, solid phase microsequencing reactions have been developed, for which either the oligonucleotide microsequencing primers or the PCR-amplified products derived from the DNA fragment of interest are immobilized. For example, immobilization can be carried out via an interaction between biotinylated DNA and streptavidin-coated microtitration wells or avidin-coated polystyrene particles.

As a further alternative, the PCR reaction generating the amplicons to be genotyped can be performed directly in solid phase conditions, following procedures such as those described in WO 96/13609, the disclosure of which is incorporated herein by reference in its entirety.

In such solid phase microsequencing reactions, incorporated ddNTPs can either be radiolabeled (see Syvänen, Clin. Chim. Acta. 226:225-236 (1994), the disclosure of which is incorporated herein by reference in its entirety) or linked to fluorescein (see Livak and Hainer, Hum. Metat. 3:379-385 (1994), the disclosure of which is incorporated herein by reference in its entirety). The detection of radiolabeled ddNTPs can be achieved through scintillation-based techniques. The detection of fluorescein-linked ddNTPs can be based on the binding of antifluorescein antibody conjugated with alkaline phosphatase, followed by incubation with a chromogenic substrate (such as p-nitrophenyl phosphate).

Other possible reporter-detection couples for use in the above microsequencing procedures include:

ddNTP linked to dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (see Harju et al., Clin Chem:39(11Pt 1):2282-2287 (1993), incorporated herein by reference in its entirety)

biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate (see WO 92/15712, incorporated herein by reference in its entirety).

A diagnosis kit based on fluorescein-linked ddNTP with antifluorescein antibody conjugated with alkaline phosphatase has been commercialized under the name PRONTO by GamidaGen Ltd.

As yet another alternative microsequencing procedure, Nyren et al. (Anal. Biochem. 208:171-175 (1993), the disclosure of which is incorporated herein by reference in its entirety) have described a solid-phase DNA sequencing procedure that relies on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA). In this procedure, the PCR-amplified products are biotinylated and immobilized on beads. The microsequencing primer is annealed and four aliquots of this mixture are separately incubated with DNA polymerase and one of the four different ddNTPs. After the reaction, the resulting fragments are washed and used as substrates in a primer extension reaction with all four dNTPs present. The progress of the DNA-directed polymerization reactions is monitored with the ELIDA. Incorporation of a ddNTP in the first reaction prevents the formation of pyrophosphate during the subsequent dNTP reaction. In contrast, no ddNTP incorporation in the first reaction gives extensive pyrophosphate release during the dNTP reaction and this leads to generation of light throughout the ELIDA reactions. From the ELIDA results, the identity of the first base after the primer is easily deduced.

It will be appreciated that several parameters of the above-described microsequencing procedures may be successfully modified by those skilled in the art without undue experimentation. In particular, high throughput improvements to these procedures may be elaborated, following principles such as those described further below.

Example 9 Sequence Analysis

DNA sequences, such as BAC inserts, containing the region carrying the candidate gene associated with the detectable trait are sequenced and their sequence is analyzed using automated software which eliminates repeat sequences while retaining potential gene sequences. The potential gene sequences are compared to numerous databases to identify potential exons using a set of scoring algorithms such as trained Hidden Markov Models, statistical analysis models (including promoter prediction tools) and the GRAIL neural network. Preferred databases for use in this analysis, the construction and use of which are further detailed in Example 17, include the following:

NRPU (Non-Redundant Protein-Unique) database: NRPU is a non-redundant merge of the publicly available NBRF/PIR, Genpept, and SwissProt databases. Homologies found with NRPU allow the identification of regions potentially coding for already known proteins or related to known proteins (translated exons).

NREST (Non-Redundant EST database): NREST is a merge of the EST subsection of the publicly available GenBank database. Homologies found with NREST allow the location of potentially transcribed regions (translated or non-translated exons).

NRN (Non-Redundant Nucleic acid database): NRN is a merge of GenBank, EMBL and their daily updates.

Any sequence giving a positive hit with NRPU, NREST or an “excellent” score using GRAIL or/and other scoring algorithms is considered a potential functional region, and is then considered a candidate for genomic analysis.

While this first screening allows the detection of the “strongest” exons, a semi-automatic scan is further applied to the remaining sequences in the context of the sequence assembly. That is, the sequences neighboring a 5′ site or an exon are submitted to another round of bioinformatics analysis with modified parameters. In this way, new exon candidates are generated for genomic analysis.

Using the above procedures, genes associated with detectable traits may be identified.

Example 10 YAC Contig Construction in the Candidate Genomic Region

Substantial amounts of LOH data supported the hypothesis that genes associated with distinct cancer types are located within a particular region of the human genome. More specifically, this region was likely to harbor a gene associated with prostate cancer.

Association studies were performed as described below in order to identify this prostate cancer gene. First, a YAC contig which contains the candidate genomic region was constructed as follows. The CEPH-Genethon YAC map for the entire human genome (Chumakov et al. (1995), supra) was used for detailed contig building in the genomic region containing genetic markers known to map in the candidate genomic region. Screening data available for several publicly available genetic markers were used to select a set of CEPH YACs localized within the candidate region. This set of YACs was tested by PCR with the above mentioned genetic markers as well as with other publicly available markers supposedly located within the candidate region. As a result of these studies, a YAC STS contig map was generated around genetic markers known to map in this genomic region. Two CEPH YACs were found to constitute a minimal tiling path in this region, with an estimated size of ca. 2 Megabases.

During this mapping effort, several publicly known STS markers were precisely located within the contig.

Example 11 below describes the identification of sets of biallelic markers within the candidate genomic region.

Example 11 BAC Contig Construction and Biallelic Markers Isolation Within the Candidate Chromosomal Region

Next, a BAC contig covering the candidate genomic region was constructed as follows. BAC libraries were obtained as described in Woo et al., Nucleic Acids Res. 22:4922-4931 (1994), the disclosure of which is incorporated herein by reference in its entirety. Briefly, the two whole human genome BamHI and HindIII libraries already described in related WIPO application No. PCT/IB98/00193 were constructed using the pBe1oBAC11 vector (Kim et al. (1996), supra).

The BAC libraries were then screened with all of the above mentioned STSs, following the procedure described in Example 1 above.

The ordered BACs selected by STS screening and verified by FISH, were assembled into contigs and new markers were generated by partial sequencing of insert ends from some of them. These markers were used to fill the gaps in the contig of BAC clones covering the candidate chromosomal region having an estimated size of 2 megabases.

FIG. 9 illustrates a minimal array of overlapping clones which was chosen for further studies, and the positions of the publicly known STS markers along said contig.

Selected BAC clones from the contig were subcloned and sequenced, essentially following the procedures described in related WIPO application No. PCT/IB98/00193.

Biallelic markers lying along the contig were identified following the processes described in related WIPO application No. PCT/IB98/00193, the disclosure of which is incorporated herein by reference in its entirety.

FIG. 9 shows the locations of the biallelic markers along the BAC contig. This first set of markers corresponds to a medium density map of the candidate locus, with an inter-marker distance averaging 50 kb-150 kb.

A second set of biallelic markers was then generated as described above in order to provide a very high-density map of the region identified using the first set of markers which can be used to conduct association studies, as explained below. This very high density map has markers spaced on average every 2-50 kb.

The biallelic markers were then used in association studies. DNA samples were obtained from individuals suffering from prostate cancer and unaffected individuals as described in Example 12.

Example 12 Collection of DNA Samples from Affected and Non-affected Individuals

Prostate cancer patients were recruited according to clinical inclusion criteria based on pathological or radical prostatectomy records. Control cases included in this study were both ethnically- and age-matched to the affected cases; they were checked for both the absence of all clinical and biological criteria defining the presence or the risk of prostate cancer, and for the absence of related familial prostate cancer cases. Both affected and control individuals were all unrelated.

The two following groups of independent individuals were used in the association studies. The first group, comprising individuals suffering from prostate cancer, contained 185 individuals. Of these 185 cases of prostate cancer, 47 cases were sporadic and 138 cases were familial. The control group contained 104 non-diseased individuals.

Haplotype analysis was conducted using additional diseased (total samples: 281) and control samples (total samples: 130), from individuals recruited according to similar criteria.

DNA was extracted from peripheral venous blood of all individuals as described in related WIPO application No. PCT/IB98/00193.

The frequencies of the biallelic markers in each population were determined as described in Example 13.

Example 13 Genotyping Affected and Control Individuals

Genotyping was performed using the following microsequencing procedure. Amplification was performed on each DNA sample using primers designed as previously explained. The pairs of primers of SEQ ID Nos.: 7849 to 7860 and 11780 to 11791 were used to generate amplicons harboring the biallelic markers of SEQ ID Nos: 3915 to 3926 or the sequences complementary thereto (markers 99-123-381, 4-26-29, 4-14-240, 4-77-151, 99-217-277, 4-67-40, 99-213-164, 99-221-377, 99-135-196, 99-1482-32, 4-73-134, and 4-65-324) using the protocols described in related WIPO application No. PCT/IB98/00193.

Microsequencing primers were designed for each of the biallelic markers, as previously described. After purification of the amplification products, the microsequencing reaction mixture was prepared by adding, in a 20 μl final volume: 10 pmol microsequencing oligonucleotide, 1 U Thermosequenase (Amersham E79000G), 1.25 μl Thermosequenase buffer (260 mM Tris HCl pH 9.5, 65 mM MgCl₂), and the two appropriate fluorescent ddNTPs (Perkin Elmer, Dye Terminator Set 401095) complementary to the nucleotides at the polymorphic site of each biallelic marker tested, following the manufacturer's recommendations. After 4 minutes at 94° C., 20 PCR cycles of 15 sec at 55° C., 5 sec at 72° C., and 10 sec at 94° C. were carried out in a Tetrad PTC-225 thermocycler (MJ Research). The unincorporated dye terminators were then removed by ethanol precipitation. Samples were finally resuspended in formamide-EDTA loading buffer and heated for 2 min at 95° C. before being loaded on a polyacrylamide sequencing gel. The data were collected by an ABI PRISM 377 DNA sequencer and processed using the GENESCAN software (Perkin Elmer).

Following gel analysis, data were automatically processed with software that allows the determination of the alleles of biallelic markers present in each amplified fragment.

The software evaluates such factors as whether the intensities of the signals resulting from the above microsequencing procedures are weak, normal, or saturated, or whether the signals are ambiguous. In addition, the software identifies significant peaks (according to shape and height criteria). Among the significant peaks, peaks corresponding to the targeted site are identified based on their position. When two significant peaks are detected for the same position, each sample is categorized as homozygous or heterozygous based on the height ratio.

Association analyzes were then performed using the biallelic markers as described below.

Example 14 Association Analysis

Association studies were run in two successive steps. In a first step, a rough localization of the candidate gene was achieved by determining the frequencies of the biallelic markers of FIG. 9 in the affected and unaffected populations. The results of this rough localization are shown in FIG. 10. This analysis indicated that a gene responsible for prostate cancer was located near the biallelic marker designated 4-67.

In a second phase of the analysis, the position of the gene responsible for prostate cancer was further refined using the very high density set of markers including the markers of SEQ ID Nos: 3915 to 3926 or the sequences complementary thereto (markers 99-123-381, 4-26-29, 4-14-240, 4-77-151, 99-217-277, 4-67-40, 99-213-164, 99-221-377, 99-135-196, 99-1482-32, 4-73-134, and 4-65-324).

As shown in FIG. 11, the second phase of the analysis confirmed that the gene responsible for prostate cancer was near the biallelic marker designated 4-67-40, most probably within a ca. 150 kb region comprising the marker.

A haplotype analysis was also performed as described in Example 15.

Example 15 Haplotype Analysis

The allelic frequencies of each of the alleles of biallelic markers 99-123-381, 4-26-29, 4-14-240, 4-77-151, 99-217-277, 4-67-40, 99-213-164, 99-221-377, and 99-135-196 were determined in the affected and unaffected populations. Table 4 lists the internal identification numbers of the markers used in the haplotype analysis (SEQ ID Nos: 3915-3923), the alleles of each marker, the most frequent allele in both unaffected individuals and individuals suffering from prostate cancer, the least frequent allele in both unaffected individuals and individuals suffering from prostate cancer, and the frequencies of the least frequent alleles in each population.

TABLE 4 Frequency of least frequent allele ** Markers Polymorphic base * Cases Controls 99-123-381 C/T 0.35 0.3  4-26-29 A/G 0.39 0.45 4-14-240 C/T 0.35 0.41 4-77-151 C/G 0.33 0.24 99-217-277 C/T 0.31 0.23 4-67-40 C/T 0.26 0.16 99-213-164 T/C 0.45 0.38 99-221-377 C/A 0.43 0.43 99-135-196 A/G 0.25 0.3  * most frequent allele/least frequent allele ** standard deviations 0.023 to 0.031 for controls; 0.018 to 0.021 for cases

Among all the theoretical potential different haplotypes based on 2 to 9 markers, 11 haplotypes showing a strong association with prostate cancer were selected. The results of these haplotype analyzes are shown in FIG. 12.

FIGS. 11 and 12 aggregate association analysis results with sequencing results—generated following the procedures further described in Example 16, which permitted the physical order and the distance between markers to be estimated.

The significance of the values obtained in FIG. 12 are underscored by the following results of computer simulations. For the computer simulations, the data from the affected individuals and the unaffected controls were pooled and randomly allocated to two groups which contained the same number of individuals as the affected and unaffected groups used to compile the data summarized in FIG. 12. A haplotype analysis was run on these artificial groups for the six markers included in haplotype 5 of FIG. 12. This experiment was reiterated 100 times and the results are shown in FIG. 13. Among 100 iterations, only 5% of the obtained haplotypes are present with a p-value less significant than E-04 as compared to the p-value of 9E-07 for haplotype 5 of FIG. 12. Furthermore, for haplotype 5 of FIG. 12, only 6% of the obtained haplotypes have a significance level below 5^(E-)03, while none of them show a significance level below 5E-03.

Thus, using the data of FIG. 13 and evaluating the associations for single marker alleles or for haplotypes will permit estimation of the risk a corresponding carrier has to develop prostate cancer. It will be appreciated that significance thresholds of relative risks will be more finely assessed according to the population tested.

Diagnostic techniques for determining an individual's risk of developing prostate cancer may be implemented as described below for the markers in the maps of the present invention, including the markers of SEQ ID Nos: 3915 to 3923 (markers 99-123-381, 4-26-29, 4-14-240, 4-77-151, 99-217-277, 4-67-40, 99-213-164, 99-221-377, and 99-135-196).

The above haplotype analysis indicated that 171 kb of genomic DNA between biallelic markers 4-14-240 and 99-221-377 totally or partially contains a gene responsible for prostate cancer. Therefore, the protein coding sequences lying within this region were characterized to locate the gene associated with prostate cancer. This analysis, described in further detail below, revealed a single protein coding sequence in the 171 kb genomic region, which was designated as the PG1 gene.

Example 16 Identification of the Genomic Sequence in the Candidate Region

Template DNA for sequencing the PG1 gene was obtained as follows. BACs E and F from FIG. 9 were subcloned as previously described. Plasmid inserts were first amplified by PCR on PE 9600 thermocyclers (Perkin-Elmer), using appropriate primers, AmpliTaqGold (Perkin-Elmer), dNTms (Boehringer), buffer and cycling conditions as recommended by the Perkin-Elmer Corporation.

PCR products were then sequenced using automatic ABI Prism 377 sequencers (Perkin Elmer, Applied Biosystems Division, Foster City, Calif.). Sequencing reactions were performed using PE 9600 thermocyclers (Perkin Elmer) with standard dye-primer chemistry and ThermoSequenase (Amersham Life Science). The primers were labeled with the JOE, FAM, ROX and TAMRA dyes. The dNTPs and ddNTPs used in the sequencing reactions were purchased from Boehringer. Sequencing buffer, reagent concentrations and cycling conditions were as recommended by Amersham.

Following the sequencing reaction, the samples were precipitated with EtOH, resuspended in formamide loading buffer, and loaded on a standard 4% acrylamide gel. Electrophoresis was performed for 2.5 hours at 3000V on an ABI 377 sequencer, and the sequence data were collected and analyzed using the ABI Prism DNA Sequencing Analysis Software, version 2.1.2.

The sequence data obtained as described above were transferred to a proprietary database, where quality control and validation steps were performed. A proprietary base-caller flagged suspect peaks, taking into account the shape of the peaks, the inter-peak resolution, and the noise level. The proprietary base-caller also performed an automatic trimming. Any stretch of 25 or fewer bases having more than 4 suspect peaks was considered unreliable and was discarded.

The sequence fragments from BAC subclones isolated as described above were assembled using Gap4 software from R. Staden (Bonfield et al. 1995). This software allows the reconstruction of a single sequence from sequence fragments. The sequence deduced from the alignment of different fragments is called the consensus sequence. Directed sequencing techniques (primer walking) were used to complete sequences and link contigs.

Potential functional sequences were then identified as described in Example 17.

Example 17 Identification of Functional Sequences

Potential exons in BAC-derived human genomic sequences were located by homology searches on protein, nucleic acid and EST (Expressed Sequence Tags) public databases. Main public databases were locally reconstructed as mentioned in Example 9. The protein database, NRPU (Non-redundant Protein Unique) is formed by a non-redundant fusion of the Genpept (Benson et al., Nucleic Acids Res. 24:1-5 (1996), the disclosure of which is incorporated herein by reference in its entirety), Swissprot (Bairoch, A. and Apweiler, R., Nucleic Acids Res. 24:21-25 (1996), the disclosure of which is incorporated herein by reference in its entirety) and PIR/NBRF (George et al., Nucleic Acids Res. 24:17-20 (1996), the disclosure of which is incorporated herein by reference in its entirety) databases. Redundant data were eliminated by using the NRDB software (Benson et al. (1996), supra) and internal repeats were masked with the XNU software (Benson et al., supra). Homologies found using the NRPU database allowed the identification of sequences corresponding to potential coding exons related to known proteins.

The EST local database is composed by the gbest section (1-9) of GenBank (Benson et al. (1996), supra), and thus contains all publicly available transcript fragments. Homologies found with this database allowed the localization of potentially transcribed regions.

The local nucleic acid database contained all sections of GenBank and EMBL (Rodriguez-Tome et al., Nucleic Acids Res. 24:6-12 (1996), the disclosure of which is incorporated herein by reference in its entirety) except the EST sections. Redundant data were eliminated as previously described.

Similarity searches in protein or nucleic acid databases were performed using the BLAST software (Altschul et al., J. Mol. Biol. 215:403-410 (1990), the disclosure of which is incorporated herein by reference in its entirety). Alignments were refined using the Fasta software, and multiple alignments used Clustal W. Homology thresholds were adjusted for each analysis based on the length and the complexity of the tested region, as well as on the size of the reference database.

Potential exon sequences identified as above were used as probes to screen cDNA libraries. Extremities of positive clones were sequenced and the sequence stretches were positioned on the genomic sequence determined above. Primers were then designed using the results from these alignments in order to enable the cloning of cDNAs derived from the gene associated with prostate cancer that was identified using the above procedures.

The obtained cDNA molecules were then sequenced and results of Northern blot analysis of prostate mRNAs supported the existence of a major cDNA having a 5-6 kb length. The structure of the gene associated with prostate cancer was evaluated as described in Example 18.

Example 18 Analysis of Gene Structure

The intron/exon structure of the gene was finally completely deduced by aligning the mRNA sequence from the cDNA obtained as described above and the genomic DNA sequence obtained as described above. This alignment permitted the determination of the positions of the introns and exons, the positions of the start and end nucleotides defining each of the at least 8 exons, the locations and phases of the 5′ and 3′ splice sites, the position of the stop codon, and the position of the polyadenylation site to be determined in the genomic sequence. This analysis also yielded the positions of the coding region in the mRNA, and the locations of the polyadenylation signal and polyA stretch in the mRNA.

The gene identified as described above comprises at least 8 exons and spans more than 52 kb. A G/C rich putative promoter region was identified upstream of the coding sequence. A CCAAT in the putative promoter was also identified. The promoter region was identified as described in Prestridge, D. S., Predicting Pol II Promoter Sequences Using Transcription Factor Binding Sites, J. Mol. Biol. 249:923-932 (1995), the disclosure of which is incorporated herein by reference in its entirety.

Additional analysis using conventional techniques, such as a 5′RACE reaction using the Marathon-Ready human prostate cDNA kit from Clontech (Catalog. No. PT1156-1), may be performed to confirm that the 5′ of the cDNA obtained above is the authentic 5′ end in the mRNA.

Alternatively, the 5′ sequence of the transcript can be determined by conducting a PCR amplification with a series of primers extending from the 5′ end of the identified coding region.

Example 19 Detection of Biallelic Markers in the Candidate Gene: DNA Extraction

Donors were unrelated and healthy. They presented a sufficient diversity for being representative of a French heterogeneous population. The DNA from 100 individuals was extracted and tested for the detection of the biallelic markers.

30 ml of peripheral venous blood were taken from each donor in the presence of EDTA. Cells (pellet) were collected after centrifugation for 10 minutes at 2000 rpm. Red cells were lysed by a lysis solution (50 ml final volume: 10 mM Tris pH7.6; 5 mM MgCl_(2; 10) mM NaCl). The solution was centrifuged (10 minutes, 2000 rpm) as many times as necessary to eliminate the residual red cells present in the supernatant, after resuspension of the pellet in the lysis solution.

The pellet of white cells was lysed overnight at 42° C. with 3.7 ml of lysis solution composed of:

3 ml TE 10-2 (Tris-HCl 10 mM, EDTA 2 mM)/NaCl 0.4 M

200 μl SDS 10%

500 μl K-proteinase (2 mg K-proteinase in TE 10-2/NaCl 0.4 M).

For the extraction of proteins, 1 ml saturated NaCl (6M) (1/3.5 v/v) was added. After vigorous agitation, the solution was centrifuged for 20 minutes at 10000 rpm. For the precipitation of DNA, 2 to 3 volumes of 100% ethanol were added to the previous supernatant, and the solution was centrifuged for 30 minutes at 2000 rpm. The DNA solution was rinsed three times with 70% ethanol to eliminate salts, and centrifuged for 20 minutes at 2000 rpm. The pellet was dried at 37° C., and resuspended in 1 ml TE 10-1 or 1 ml water. The DNA concentration was evaluated by measuring the OD at 260 nm (1 unit OD=50 μg/ml DNA).

To determine the presence of proteins in the DNA solution, the OD 260/OD 280 ratio was determined. Only DNA preparations having a OD 260/OD 280 ratio between 1.8 and 2 were used in the subsequent examples described below.

The pool was constituted by mixing equivalent quantities of DNA from each individual.

Example 20 Detection of the Biallelic Markers: Amplification of Genomic DNA by PCR

The amplification of specific genomic sequences of the DNA samples of Example 19 was carried out on the pool of DNA obtained previously using the amplification primers of SEQ ID Nos: 7861 to 7865 and 11792 to 11796. In addition, 50 individual samples were similarly amplified.

PCR assays were performed using the following protocol:

Final volume 25 μl DNA 2 ng/μl MgCl2 2 mM dNTP (each) 200 μM primer (each) 2.9 ng/μl Ampli Taq Gold DNA polymerase 0.05 unit/μl PCR buffer (10× = 0.1 M TrisHCl pH 8.3 0.5 M KCl 1×

Pairs of first primers were designed to amplify the promoter region, exons, and 3′ end of the candidate asthma-associated gene using the sequence information of the candidate gene and the OSP software (Hillier & Green, 1991). These first primers were about 20 nucleotides in length and contained a common oligonucleotide tail upstream of the specific bases targeted for amplification which was useful for sequencing. The synthesis of these primers was performed following the phosphoramidite method, on a GENSET UFPS 24.1 synthesizer.

DNA amplification was performed on a Genius II thermocycler. After heating at 94° C. for 10 min, 40 cycles were performed. Each cycle comprised: 30 sec at 94° C., 55° C. for 1 min, and 30 sec at 72° C. For final elongation, 7 min at 72° C. ended the amplification. The quantities of the amplification products obtained were determined on 96-well microtiter plates, using a fluorometer and Picogreen as intercalant agent (Molecular Probes).

Example 21 Detection of the Biallelic Markers Sequencing of Amplified Genomic DNA and Identification of Polymorphisms

The sequencing of the amplified DNA obtained in Example 20 was carried out on ABI 377 sequencers. The sequences of the amplification products were determined using automated dideoxy terminator sequencing reactions with a dye terminator cycle sequencing protocol. The products of the sequencing reactions were run on sequencing gels and the sequences were analyzed as formerly described.

The sequence data were further evaluated using the above mentioned polymorphism analysis software designed to detect the presence of biallelic markers among the pooled amplified fragments. The polymorphism search was based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position as described previously.

Six fragments of amplification were analyzed. In these segments, 8 biallelic markers were detected (SEQ ID Nos: 3927 to 3934). The localization of the biallelic markers, the polymorphic bases of each allele, and the frequencies of the most frequent alleles was as shown in Table 5.

TABLE 5 Locali- Amp- Marker Origin of zation licon Name DNA gene Polymorphism Frequency 1 10-204-326 Ind. Promoter A/G 96.2 (G) 2 10-32-357 Pool Intron 1 A/C 67.7 (C) 3 10-33-175 Ind. Exon 2 C/T 97.3 (C) 3 10-33-234 Pool Intron 2 A/C 56.7 (C) 3 10-33-327 Ind. Intron 2 C/T 75.3 (T) 5 10-35-358 Pool Intron 4 C/G 67.9 (G) 5 10-35-390 Ind. Intron 4 C/T 82 (C) 6 10-36-164 Ind. Exon 5 A/G 99.5 (G)

Allelic frequencies were determined in a population of random blood donors from French Caucasian origin. Their wide range is due to the fact that, besides screening a pool of 100 individuals to generate biallelic markers as described above, polymorphism searches were also conducted in an individual testing format for 50 samples. This strategy was chosen here to provide a potential shortcut towards the identification of putative causal mutations in the association studies using them. As the 10-36-164 biallelic marker (SEQ ID No: 3933) was found in only one individual, this marker was not considered in the association studies.

The fourth fragment of amplification carrying exon 3 (not shown in the Table) was not polymorphic in the tested samples (1 pool+50 individuals).

Example 22 Validation of the Polymorphisms Through Microsequencing

The biallelic markers identified in Example 21 were further confirmed and their respective frequencies were determined through microsequencing. Microsequencing was carried out for each individual DNA sample described in Example 19.

Amplification from genomic DNA of individuals was performed by PCR as described above for the detection of the biallelic markers with the same set of PCR primers described above.

The preferred primers used in microsequencing had about 19 nucleotides in length and hybridized just upstream of the considered polymorphic base. Five primers hybridized with the non-coding strand of the gene. For the biallelic markers 10-204-326, 10-35-358 and 10-36-164, primers hybridized with the coding strand of the gene.

The microsequencing reaction was performed as described in Example 13.

Example 23 Association Study Between Asthma and the Biallelic Markers of the Candidate Gene Collection of DNA Samples from Affected and Non-Affected Individuals

The asthmatic population used to perform association studies in order to establish whether the candidate gene was an asthma-causing gene consisted of 298 individuals. More than 90% of these 298 asthmatic individuals had a Caucasian ethnic background.

The control population consisted of 373 unaffected individuals, among which 279 French (at least 70% were of Caucasian origin) and 94 American (at least 90% were of Caucasian origin).

DNA samples were obtained from asthmatic and non-asthmatic individuals as described above.

Example 24 Association Study Between Asthma and the Biallelic Markers of the Candidate Gene Genotyping of Affected and Control Individuals

The general strategy to perform the association studies was to individually scan the DNA samples from all individuals in each of the populations described above in order to establish the allele frequencies of the above described biallelic markers in each of these populations.

Allelic frequencies of the above-described biallelic markers in each population were determined by performing microsequencing reactions on amplified fragments obtained by genomic PCR performed on the DNA samples from each individual. Genomic PCR and microsequencing were performed as detailed above in Examples 20 and 22 using the described amplification and microsequencing primers.

Example 25 Association study Between Asthma and the Biallelic Markers of the Candidate Gene

Table 6 shows the results of the association study between five biallelic markers in the candidate gene and asthma.

TABLE 6 Allelic frequencies (%) Asthmatics Controls Frequency Markers 298 individuals 373 individuals diff. P value 10-32-357 A 38.6 A 29.8 8.8 7.34 × 10⁻⁴ 10-33-234 A 49 A 44.3 4.7 8.86 × 10⁻² 10-33-327 T 78.5 T 74.6 3.9  1.0 × 10⁻¹ 10-35-358 G 72.3 G 66.9 5.4 3.59 × 10⁻² 10-35-390 T 30.4 T 20.3 10.1 2.33 × 10⁻⁵

As shown in Table 6, markers 10-32-357 and 10-35-390 presented a strong association with asthma, this association being highly significant (p value=7.34×10−4 for marker 10-32-357 and 2.33×10−5 for marker 10-35-390).

Three markers showed moderate association when tested independently, namely 10-33-234, 10-33-327,10-35-358.

It is worth mentioning that allelic frequencies for each of the biallelic markers of Table 7 were separately measured within the French control population (279 individuals) and the American control population (94 individuals). The differences in allele frequencies between the two populations were between 1% and 7%, with p-values above 10-1. These data confirmed that the combined French/American control population (373 individuals) was homogeneous enough to be used as a control population for the present association study.

Example 26 Association Studies: Haplotype Frequency Analysis

As already shown, one way of increasing the statistical power of individual markers, is by performing haplotype association analysis. A haplotype analysis for association of markers in the candidate gene and asthma was performed by estimating the frequencies of all possible haplotypes for biallelic markers 10-32-357, 10-33-234, 10-33-327, 10-35-358 and 10-35-390 in the asthmatic and control populations described in Example 25 (Table 6), and comparing these frequencies by means of a chi square statistical test (one degree of freedom). Haplotype estimations were performed by applying the Expectation-Maximization (EM) algorithm (Excoffier L & Slatkin M, 1995, Mol.Biol.Evol. 12:921-927, the disclosure of which is incorporated herein by reference in its entirety), using the EM-HAPLO program (Hawley Ma., Pakstis A J & Kidd K K, 1994, Am.J.Phys.Anthropol. 18:104, the disclosure of which is incorporated herein by reference in its entirety).

The results of such haplotype analysis are shown in Table 7.

TABLE 7 Haplotype frequencies Odds Markers 10-32-357 10-33-234 10-33-327 10-35-358 10-35-390 Asthm. Controls ratio P value Frequency 8.8 4.7 3.9 5.4 10.1 diff. P value 7.34 × 10⁻⁴ 8.86 × 10⁻² 1.0 × 10⁻¹ 3.59 × 10⁻² 2.33 × 10⁻⁵ Haplotype 1 A T 0.2 0.11 2.02 8.47 × 10⁻⁶ 5 Haplotype 2 A T G 0.27 0.18 1.68 2.81 × 10⁻⁴ Haplotype 3 A A T G T 0.18 0.09 2.22 3.95 × 10⁻⁵

A two-marker haplotype covering markers 10-32-357 and 10-35-390 (haplotype 1, AT alleles respectively) presented a p value of 8.47×10−6, an odds ratio of 2.02 and haplotype frequencies of 0.2 for asthmatic and 0.11 for control populations respectively.

A three-marker haplotype covering markers 10-33-234, 10-33-327 and 10-35-358 (haplotype 2, ATG alleles respectively) presented a p value of 2.81×10−4, an odds ratio of 1.68 and haplotype frequencies of 0.27 for asthmatic and 0.18 for control populations respectively.

A five-marker haplotype covering markers 10-32-357, 10-33-234, 10-33-327, 10-35-358 and 10-35-390 (haplotype 3, AATGT alleles respectively) presented a p value of 3.95×10−5, an odds ratio of 2.22 and haplotype frequencies of 0.18 for asthmatic and 0.09 for control populations respectively.

Haplotype association analysis thus increased the statistical power of the individual marker association studies when compared to single-marker analysis (from p values between 10⁻¹ and 2×10⁻⁵ for the individual markers to p values between 3×10⁻⁴ and 8×10⁻⁶ for the three-marker haplotype, haplotype 2).

The significance of the values obtained for the haplotype association analysis was evaluated by the following computer simulation test. The genotype data from the asthmatic and control individuals were pooled and randomly allocated to two groups which contained the same number of individuals as the trait-positive and trait-negative groups used to produce the data summarized in Table 7. A haplotype analysis was then run on these artificial groups for the three haplotypes presented in Table 6. This experiment was reiterated 1000 times and the results are shown in Table 8.

TABLE 8 Permutation Test Chi-Square Maximal Haplotype Average Chi-Square Chi-Square P value Haplotype 1 19.70 1.2 11.6 1.0 × 10⁻³ (A---T) Haplotype 2 13.49 1.2 10.5 1.0 × 10⁻³ (-ATG-) Haplotype 3 16.66 1.2 9.3 1.0 × 10⁻³ (AATGT)

The results in Table 8 show that among 1000 iterations only 1‰ of the obtained haplotypes has a p value comparable to the one obtained in Table 4.

These results clearly validate the statistical significance of the haplotypes obtained (haplotypes 1, 2 and 3, Table 7).

Example 27 Extraction of DNA

30 ml of blood are taken from the individuals in the presence of EDTA. Cells (pellet) are collected after centrifugation for 10 minutes at 2000 rpm. Red cells are lysed by a lysis solution (50 ml final volume: 10 mM Tris pH7.6; 5 mM MgCl₂; 10 mM NaCl). The solution is centrifuged (10 minutes, 2000 rpm) as many times as necessary to eliminate the residual red cells present in the supernatant, after resuspension of the pellet in the lysis solution.

The pellet of white cells is lysed overnight at 42° C. with 3.7 ml of lysis solution composed of:

3 ml TE 10-2 (Tris-HCl 10 mM, EDTA 2 mM)/NaCl 0.4 M

200 μl SDS 10%

500 μl K-proteinase (2 mg K-proteinase in TE 10-2/NaCl 0.4 M).

For the extraction of proteins, 1 ml saturated NaCl (6M) (1/3.5 v/v) is added. After vigorous agitation, the solution is centrifuged for 20 minutes at 10000 rpm. For the precipitation of DNA, 2 to 3 volumes of 100% ethanol are added to the previous supernatant, and the solution is centrifuged for 30 minutes at 2000 rpm. The DNA solution is rinsed three times with 70% ethanol to eliminate salts, and centrifuged for 20 minutes at 2000 rpm. The pellet is dried at 37° C., and resuspended in 1 ml TE 10-1 or 1 ml water. The DNA concentration is evaluated by measuring the OD at 260 nm (1 unit OD=50 μg/ml DNA).

To evaluate the presence of proteins in the DNA solution, the OD 260/OD 280 ratio is determined. Only DNA preparations having a OD 260/OD 280 ratio between 1.8 and 2 are used in the subsequent steps described below.

Once genomic DNA from every individual in the given population has been extracted, it is preferred that a fraction of each DNA sample is separated, after which a pool of DNA is constituted by assembling equivalent DNA amounts of the separated fractions into a single one.

Although this invention has been described in terms of certain preferred embodiments, other embodiments which will be apparent to those of ordinary skill in the art of view of the disclosure herein are also within the scope of this invention. Accordingly, the scope of the invention is intended to be defined only by reference to the appended claims.

TABLE 1 SEQ Preferred Amplification primer ID Allele microseq. Upstream Downstream No. Marker Name 1^(ST) 2^(ND) primer (PU) (RP) 1 99-109-224 G C S 3935 7866 2 99-1126-384 A G S 3936 7867 3 99-114-68 G C S 3937 7868 4 99-1151-516 A C S 3938 7869 5 99-1165-159 C T S 3939 7870 6 99-1167-201 A G A 3940 7871 7 99-117-205 C T S 3941 7872 8 99-118-92 C T S 3942 7873 9 99-1217-332 C T A 3943 7874 10 99-1233-183 A G S 3944 7875 11 99-12478-263 G T A 3945 7876 12 99-12487-301 A C S 3946 7877 13 99-12497-155 C T S 3947 7878 14 99-12503-44 G C S 3948 7879 15 99-12504-402 A T S 3949 7880 16 99-12505-374 A G A 3950 7881 17 99-12506-199 G T A 3951 7882 18 99-12509-423 C T S 3952 7883 19 99-12513-146 G C S 3953 7884 20 99-12514-170 G C S 3954 7885 21 99-12515-205 G C S 3955 7886 22 99-12516-524 A G A 3956 7887 23 99-12518-325 C T S 3957 7888 24 99-12523-255 C T S 3958 7889 25 99-12525-277 C T S 3959 7890 26 99-12526-317 C T S 3960 7891 27 99-12527-292 A G A 3961 7892 28 99-12531-30 C T S 3962 7893 29 99-12532-199 A T S 3963 7894 30 99-12534-207 A C S 3964 7895 31 99-12535-362 A C S 3965 7896 32 99-12537-340 G C S 3966 7897 33 99-12538-142 A C S 3967 7898 34 99-12539-287 C T S 3968 7899 35 99-12540-426 C T S 3969 7900 36 99-12541-307 C T S 3970 7901 37 99-12545-121 A G A 3971 7902 38 99-12548-88 A G A 3972 7903 39 99-12558-167 C T S 3973 7904 40 99-12562-291 C T S 3974 7905 41 99-12564-354 A T S 3975 7906 42 99-12565-273 C T S 3976 7907 43 99-12575-248 G C S 3977 7908 44 99-12576-325 C T S 3978 7909 45 99-12580-268 A G A 3979 7910 46 99-12585-85 A C S 3980 7911 47 99-12593-103 A C S 3981 7912 48 99-12600-283 G C S 3982 7913 49 99-12608-71 C T S 3983 7914 50 99-12610-106 C T S 3984 7915 51 99-12611-311 G T A 3985 7916 52 99-12613-366 A G A 3986 7917 53 99-12615-235 A C S 3987 7918 54 99-12617-412 G C S 3988 7919 55 99-12618-211 C T S 3989 7920 56 99-12619-367 A G A 3990 7921 57 99-12621-114 A G A 3991 7922 58 99-12624-61 A T S 3992 7923 59 99-1263-276 A G S 3993 7924 60 99-12632-165 C T S 3994 7925 61 99-12637-62 C T S 3995 7926 62 99-12639-311 G C S 3996 7927 63 99-12640-179 C T A 3997 7928 64 99-12650-200 C T A 3998 7929 65 99-12651-297 G C S 3999 7930 66 99-12652-459 A G S 4000 7931 67 99-12654-278 G T A 4001 7932 68 99-12656-303 C T A 4002 7933 69 99-12658-206 C T A 4003 7934 70 99-12661-92 G T A 4004 7935 71 99-12668-329 C T A 4005 7936 72 99-1268-177 A G A 4006 7937 73 99-12733-366 G C S 4007 7938 74 99-12738-57 G C S 4008 7939 75 99-12740-354 C T A 4009 7940 76 99-12749-286 A G S 4010 7941 77 99-12750-369 A T S 4011 7942 78 99-12751-406 C T A 4012 7943 79 99-12755-421 A G S 4013 7944 80 99-12756-344 A C S 4014 7945 81 99-12757-240 A G S 4015 7946 82 99-12759-420 G T A 4016 7947 83 99-12777-71 A G S 4017 7948 84 99-12782-76 A C S 4018 7949 85 99-12794-299 G C S 4019 7950 86 99-128-60 C T S 4020 7951 87 99-12816-101 G C S 4021 7952 88 99-12817-358 C T A 4022 7953 89 99-12819-165 A G S 4023 7954 90 99-12826-408 A T S 4024 7955 91 99-12831-345 A C S 4025 7956 92 99-12836-387 C T A 4026 7957 93 99-12842-305 C T A 4027 7958 94 99-12843-337 A G S 4028 7959 95 99-12844-130 A G S 4029 7960 96 99-12847-37 A G S 4030 7961 97 99-12848-204 A G S 4031 7962 98 99-12852-260 A G S 4032 7963 99 99-12856-183 A C S 4033 7964 100 99-12878-291 C T S 4034 7965 101 99-12880-282 C T S 4035 7966 102 99-12884-248 A G A 4036 7967 103 99-12885-261 A C S 4037 7968 104 99-12898-364 C T S 4038 7969 105 99-12899-307 C T S 4039 7970 106 99-1290-291 C T S 4040 7971 107 99-12900-165 G C S 4041 7972 108 99-12901-316 A G A 4042 7973 109 99-12903-381 C T S 4043 7974 110 99-12907-295 A G A 4044 7975 111 99-12908-369 G C S 4045 7976 112 99-12913-197 C T S 4046 7977 113 99-12914-227 G T A 4047 7978 114 99-12924-273 G C S 4048 7979 115 99-12925487 C T S 4049 7980 116 99-12926-332 C T A 4050 7981 117 99-12931-173 A G S 4051 7982 118 99-12948-61 A T S 4052 7983 119 99-12952-199 G C S 4053 7984 120 99-12956-43 C T A 4054 7985 121 99-12957-448 C T A 4055 7986 122 99-12961-318 A G S 4056 7987 123 99-12962-181 A G S 4057 7988 124 99-12963-255 C T A 4058 7989 125 99-12964-230 C T A 4059 7990 126 99-13021-124 C T S 4060 7991 127 99-13036-313 A C S 4061 7992 128 99-13045-385 A C S 4062 7993 129 99-13051-235 A G S 4063 7994 130 99-13061-100 C T S 4064 7995 131 99-13064-328 C T S 4065 7996 132 99-13065-311 C T S 4066 7997 133 99-13070-207 G T A 4067 7998 134 99-13098-369 A G S 4068 7999 135 99-13106-251 A G S 4069 8000 136 99-13115-106 C T A 4070 8001 137 99-13121-198 A G S 4071 8002 138 99-13130-75 A G S 4072 8003 139 99-13133-341 A G S 4073 8004 140 99-13134-79 A G S 4074 8005 141 99-13165-216 A G S 4075 8006 142 99-13178-252 C T A 4076 8007 143 99-13192-272 A G S 4077 8008 144 99-13193-453 C T A 4078 8009 145 99-13201-154 C T A 4079 8010 146 99-13203-79 A G S 4080 8011 147 99-13215-109 C T A 4081 8012 148 99-13218-103 G C S 4082 8013 149 99-13219-378 A G S 4083 8014 150 99-13222-274 C T A 4084 8015 151 99-13224-351 C T S 4085 8016 152 99-13227-270 A C S 4086 8017 153 99-13229-192 G T A 4087 8018 154 99-13232-494 G C S 4088 8019 155 99-13237-44 G T S 4089 8020 156 99-13238-276 A G S 4090 8021 157 99-13241-49 C T A 4091 8022 158 99-13246-251 A G S 4092 8023 159 99-13250-439 C T A 4093 8024 160 99-13251-118 G T A 4094 8025 161 99-13258-232 G T A 4095 8026 162 99-13260-358 G C S 4096 8027 163 99-13262-376 G C S 4097 8028 164 99-13269-144 A G S 4098 8029 165 99-13270-309 G T A 4099 8030 166 99-13271-163 A G S 4100 8031 167 99-13272-151 A G S 4101 8032 168 99-13273-144 A C S 4102 8033 169 99-13276-168 C T A 4103 8034 170 99-13279-301 A G S 4104 8035 171 99-13286-58 A G S 4105 8036 172 99-13287-298 C T A 4106 8037 173 99-13294-281 A G S 4107 8038 174 99-13296-330 C T A 4108 8039 175 99-13320-352 A G S 4109 8040 176 99-13332-259 C T A 4110 8041 177 99-13334-136 A C S 4111 8042 178 99-13336-364 A G S 4112 8043 179 99-13339-335 G C S 4113 8044 180 99-13354-225 A G S 4114 8045 181 99-13368-221 C T A 4115 8046 182 99-13394-42 A G S 4116 8047 183 99-13395-110 C T A 4117 8048 184 99-13396-258 G C S 4118 8049 185 99-134-362 G T A 4119 8050 186 99-13401-106 A C A 4120 8051 187 99-13404-373 A G S 4121 8052 188 99-13406-279 G T A 4122 8053 189 99-1342-51 C T A 4123 8054 190 99-13429-188 A C S 4124 8055 191 99-13439-327 A G S 4125 8056 192 99-13443-275 C T A 4126 8057 193 99-13450-276 A G S 4127 8058 194 99-13457-138 G C S 4128 8059 195 99-1346-503 C T S 4129 8060 196 99-13462-263 A G S 4130 8061 197 99-13486-358 A G S 4131 8062 198 99-13489-396 C T A 4132 8063 199 99-13499-445 A G S 4133 8064 200 99-13502-118 C T S 4134 8065 201 99-13509-388 A G A 4135 8066 202 99-1351-264 A T S 4136 8067 203 99-13515-428 C T S 4137 8068 204 99-13525-395 C T S 4138 8069 205 99-13526-368 C T S 4139 8070 206 99-13531-449 A T S 4140 8071 207 99-13536-134 C T S 4141 8072 208 99-13540-338 G C S 4142 8073 209 99-13541-85 A G A 4143 8074 210 99-13545-215 C T S 4144 8075 211 99-13552-172 A C S 4145 8076 212 99-13553-390 A C S 4146 8077 213 99-13555-402 A G A 4147 8078 214 99-1356-500 A T S 4148 8079 215 99-13567-258 C T S 4149 8080 216 99-13586-230 G T A 4150 8081 217 99-13588-238 A C S 4151 8082 218 99-13589-362 A G A 4152 8083 219 99-1359-355 C T S 4153 8084 220 99-13591-360 G C S 4154 8085 221 99-13592-304 A C S 4155 8086 222 99-13596-69 A C S 4156 8087 223 99-13598-260 G C S 4157 8088 224 99-13600-305 A G S 4158 8089 225 99-13601-360 A G S 4159 8090 226 99-13605-208 C T A 4160 8091 227 99-13606-83 G C S 4161 8092 228 99-1362-126 A G A 4162 8093 229 99-13624-415 C T A 4163 8094 230 99-13638-354 A G S 4164 8095 231 99-13644-439 G C S 4165 8096 232 99-13647-278 C T A 4166 8097 233 99-13652-407 G C S 4167 8098 234 99-13663-218 C T A 4168 8099 235 99-13666-275 A T S 4169 8100 236 99-1367-287 A G A 4170 8101 237 99-13671-396 C T A 4171 8102 238 99-13678-251 C T A 4172 8103 239 99-13679-285 C T A 4173 8104 240 99-1368-299 C T S 4174 8105 241 99-13684-488 A C S 4175 8106 242 99-13687-316 A G S 4176 8107 243 99-1373-358 A T S 4177 8108 244 99-1376-196 A T S 4178 8109 245 99-13790-129 C T A 4179 8110 246 99-13798-284 A G S 4180 8111 247 99-13831-102 A G S 4181 8112 248 99-13832-226 C T A 4182 8113 249 99-13835-39 G C S 4183 8114 250 99-1385-91 G C S 4184 8115 251 99-13853-256 C T A 4185 8116 252 99-13854-363 C T A 4186 8117 253 99-13860-368 C T A 4187 8118 254 99-13861-227 A G S 4188 8119 255 99-13866-198 C T A 4189 8120 256 99-13868-240 C T A 4190 8121 257 99-1387-462 C T A 4191 8122 258 99-13876-55 A G S 4192 8123 259 99-13878-385 A C S 4193 8124 260 99-1388-242 A G A 4194 8125 261 99-13880-185 A G S 4195 8126 262 99-13883-103 A G S 4196 8127 263 99-13887-190 C T A 4197 8128 264 99-13888-332 C T A 4198 8129 265 99-13892-338 A G S 4199 8130 266 99-13897-431 G T A 4200 8131 267 99-1391-204 C T S 4201 8132 268 99-13912-89 C T A 4202 8133 269 99-13913-278 A G S 4203 8134 270 99-13914-169 A G S 4204 8135 271 99-1392-200 C T S 4205 8136 272 99-13920-172 A G S 4206 8137 273 99-13925-97 A G S 4207 8138 274 99-13929-201 A C S 4208 8139 275 99-13932-229 C T A 4209 8140 276 99-1394-271 A G A 4210 8141 277 99-13956-119 G C S 4211 8142 278 99-13960-142 C T A 4212 8143 279 99-13962-339 A G S 4213 8144 280 99-13980-150 C T A 4214 8145 281 99-13996-123 A G S 4215 8146 282 99-13997-181 C T A 4216 8147 283 99-13998-421 C T A 4217 8148 284 99-140-130 C T S 4218 8149 285 99-14004-328 G C S 4219 8150 286 99-14005-344 C T A 4220 8151 287 99-14009-133 C T A 4221 8152 288 99-14010-165 C T A 4222 8153 289 99-14013-125 C T A 4223 8154 290 99-14025-459 A G S 4224 8155 291 99-1404-135 A G S 4225 8156 292 99-14046-270 A G S 4226 8157 293 99-14050-295 G T A 4227 8158 294 99-14068-214 C T A 4228 8159 295 99-14072-363 C T A 4229 8160 296 99-14080-436 G T A 4230 8161 297 99-14083-346 A G S 4231 8162 298 99-14087-429 G T A 4232 8163 299 99-14090-398 A T S 4233 8164 300 99-14094-274 A T S 4234 8165 301 99-14119-101 C T S 4235 8166 302 99-14120-283 A G A 4236 8167 303 99-14127-127 A G A 4237 8168 304 99-1413-137 G C S 4238 8169 305 99-14135-375 C T S 4239 8170 306 99-14139-321 A T S 4240 8171 307 99-14140-310 A G A 4241 8172 308 99-14145-220 G C S 4242 8173 309 99-14147-369 A G A 4243 8174 310 99-14149-351 G C S 4244 8175 311 99-1416-589 A G A 4245 8176 312 99-14161-267 A G A 4246 8177 313 99-14162-180 C T S 4247 8178 314 99-14166-217 G C S 4248 8179 315 99-14175-380 A G S 4249 8180 316 99-14179-191 A G S 4250 8181 317 99-14186-424 A G S 4251 8182 318 99-14197-144 A G S 4252 8183 319 99-14203-268 A C S 4253 8184 320 99-14204-468 G T A 4254 8185 321 99-14220-351 A G S 4255 8186 322 99-1423-361 A C S 4256 8187 323 99-14250-381 A G S 4257 8188 324 99-14254-305 G T A 4258 8189 325 99-14256-133 C T A 4259 8190 326 99-1426-185 C T S 4260 8191 327 99-14260-261 C T A 4261 8192 328 99-14277-73 A G S 4262 8193 329 99-14282-334 A C S 4263 8194 330 99-14285-381 C T A 4264 8195 331 99-14286-220 G T A 4265 8196 332 99-14309-259 C T S 4266 8197 333 99-14315-405 A C S 4267 8198 334 99-14329-205 G C S 4268 8199 335 99-14331-64 A G S 4269 8200 336 99-14332-437 C T A 4270 8201 337 99-14343-408 A G S 4271 8202 338 99-14345-139 C T A 4272 8203 339 99-14356-141 A G S 4273 8204 340 99-1437-325 C T S 4274 8205 341 99-14385-117 A T S 4275 8206 342 99-14392-431 A C S 4276 8207 343 99-14393-190 C T A 4277 8208 344 99-144-392 C T S 4278 8209 345 99-14405-105 A G S 4279 8210 346 99-1442-224 G T A 4280 8211 347 99-14444-193 G C S 4281 8212 348 99-14446-337 A G S 4282 8213 349 99-14452-263 C T S 4283 8214 350 99-14459-44 G C S 4284 8215 351 99-14468-247 C T A 4285 8216 352 99-14470-243 A G S 4286 8217 353 99-14492-322 C T A 4287 8218 354 99-14497-220 A G S 4288 8219 355 99-14505-250 C T A 4289 8220 356 99-14518-57 C T A 4290 8221 357 99-1453-204 C T S 4291 8222 358 99-14553-224 C T S 4292 8223 359 99-14562-402 A G S 4293 8224 360 99-14566-320 C T A 4294 8225 361 99-14574-310 G C S 4295 8226 362 99-14581-365 C T A 4296 8227 363 99-14591-172 G C S 4297 8228 364 99-14595-210 C T A 4298 8229 365 99-14596-174 C T A 4299 8230 366 99-14597-85 G C S 4300 8231 367 99-14598-91 C T A 4301 8232 368 99-14599-220 C T A 4302 8233 369 99-14600-207 A G S 4303 8234 370 99-14601-448 C T A 4304 8235 371 99-14607-267 C T A 4305 8236 372 99-14609-467 G T A 4306 8237 373 99-14610-351 A C S 4307 8238 374 99-14611-241 G C S 4308 8239 375 99-14612-100 G C S 4309 8240 376 99-14614-248 A G S 4310 8241 377 99-14615-65 A G S 4311 8242 378 99-14616-35 A G S 4312 8243 379 99-14618-147 G C S 4313 8244 380 99-14619-325 A C S 4314 8245 381 99-1462-238 G C S 4315 8246 382 99-14620-253 C T A 4316 8247 383 99-14621-96 G C S 4317 8248 384 99-14622-276 C T A 4318 8249 385 99-14626-307 A G S 4319 8250 386 99-14627-272 C T A 4320 8251 387 99-14628-312 A C S 4321 8252 388 99-14629-274 A G S 4322 8253 389 99-14630-75 C T A 4323 8254 390 99-14634-350 A G S 4324 8255 391 99-14635-296 G C S 4325 8256 392 99-14637-366 G C S 4326 8257 393 99-14638-276 A G S 4327 8258 394 99-14643-27 C T S 4328 8259 395 99-14644-395 G C S 4329 8260 396 99-14647-227 C T A 4330 8261 397 99-14651-205 A G S 4331 8262 398 99-14652-120 A G S 4332 8263 399 99-14653-138 C T A 4333 8264 400 99-14662-352 A G S 4334 8265 401 99-14664-289 C T A 4335 8266 402 99-14665-199 A G S 4336 8267 403 99-14669-238 A G S 4337 8268 404 99-14671-175 C T A 4338 8269 405 99-14676-313 A T S 4339 8270 406 99-14677-358 G C S 4340 8271 407 99-14678-75 G C S 4341 8272 408 99-14679-241 C T A 4342 8273 409 99-1468-435 C T S 4343 8274 410 99-1469-47 G C S 4344 8275 411 99-14690-84 G T A 4345 8276 412 99-14692-46 A G S 4346 8277 413 99-14699-149 C T A 4347 8278 414 99-147-181 A G A 4348 8279 415 99-14701-264 A G S 4349 8280 416 99-14704-59 A C S 4350 8281 417 99-14708-142 G T A 4351 8282 418 99-1471-571 C T S 4352 8283 419 99-14710-107 C T A 4353 8284 420 99-14712-163 C T A 4354 8285 421 99-14714-237 C T S 4355 8286 422 99-14717-132 A G S 4356 8287 423 99-1472-435 A G A 4357 8288 424 99-14722-272 C T A 4358 8289 425 99-14729-284 A T S 4359 8290 426 99-14733-26 A T S 4360 8291 427 99-14735-328 A G S 4361 8292 428 99-1474-156 G T A 4362 8293 429 99-14746-377 A G S 4363 8294 430 99-14753-194 G T A 4364 8295 431 99-14756-270 A T S 4365 8296 432 99-1476-172 G C S 4366 8297 433 99-14761-194 A G S 4367 8298 434 99-14773-383 G T A 4368 8299 435 99-14776-79 G C S 4369 8300 436 99-14777-100 C T S 4370 8301 437 99-14782-152 G C S 4371 8302 438 99-14784-212 A G S 4372 8303 439 99-14785-92 A C S 4373 8304 440 99-14786-59 A G S 4374 8305 441 99-1479-158 C T S 4375 8306 442 99-14792-43 C T S 4376 8307 443 99-14796-227 C T A 4377 8308 444 99-14799-57 A G S 4378 8309 445 99-148-182 A G A 4379 8310 446 99-1480-290 G T A 4380 8311 447 99-14802-60 A T S 4381 8312 448 99-14803-157 C T A 4382 8313 449 99-14804-216 A G S 4383 8314 450 99-14805-58 A G S 4384 8315 451 99-14806-108 G C S 4385 8316 452 99-14807-150 G C S 4386 8317 453 99-1481-285 G T A 4387 8318 454 99-14810-407 C T A 4388 8319 455 99-14812-189 G C S 4389 8320 456 99-14817-323 C T A 4390 8321 457 99-14818-430 A G S 4391 8322 458 99-14819-278 G C S 4392 8323 459 99-14820-76 A T S 4393 8324 460 99-14821-48 C T A 4394 8325 461 99-14826-238 G T A 4395 8326 462 99-14828-214 C T A 4396 8327 463 99-14833-226 A T S 4397 8328 464 99-1484-328 G C S 4398 8329 465 99-14843-195 G T A 4399 8330 466 99-14844-143 C T A 4400 8331 467 99-1485-251 G T A 4401 8332 468 99-14850-136 A G S 4402 8333 469 99-14856-260 A G S 4403 8334 470 99-14861-387 A G S 4404 8335 471 99-14862-171 A T S 4405 8336 472 99-14865-386 A G S 4406 8337 473 99-14867-160 A T S 4407 8338 474 99-14872-326 A G S 4408 8339 475 99-14873453 C T A 4409 8340 476 99-14875-411 C T A 4410 8341 477 99-14879-398 G C S 4411 8342 478 99-14881-231 C T A 4412 8343 479 99-14882-382 G C S 4413 8344 480 99-14883-123 G C S 4414 8345 481 99-1489-76 A C S 4415 8346 482 99-14890-358 G T A 4416 8347 483 99-14892-237 A G S 4417 8348 484 99-14894-52 A G S 4418 8349 485 99-14895-343 C T A 4419 8350 486 99-14897-356 C T A 4420 8351 487 99-1490-381 C T S 4421 8352 488 99-14907-411 G T A 4422 8353 489 99-1493-280 A G A 4423 8354 490 99-14937-42 A C S 4424 8355 491 99-14939-240 A C S 4425 8356 492 99-1494-598 A G A 4426 8357 493 99-14940-224 A G S 4427 8358 494 99-14950-346 A T S 4428 8359 495 99-14959-81 A T S 4429 8360 496 99-14961-193 G C S 4430 8361 497 99-14962-120 A G S 4431 8362 498 99-14966-187 A G S 4432 8363 499 99-14970-352 A G S 4433 8364 500 99-14978-200 G T A 4434 8365 501 99-1498-120 C T S 4435 8366 502 99-14983-186 G T A 4436 8367 503 99-14984-35 C T A 4437 8368 504 99-15005-169 C T A 4438 8369 505 99-15007-369 A G S 4439 8370 506 99-1501-296 A G A 4440 8371 507 99-15016-293 C T A 4441 8372 508 99-15018-270 A G S 4442 8373 509 99-15019-408 A G S 4443 8374 510 99-15021-189 A G S 4444 8375 511 99-15030-271 A C S 4445 8376 512 99-15039-277 G T A 4446 8377 513 99-1504-252 A G A 4447 8378 514 99-15043-175 A G S 4448 8379 515 99-15046-54 C T A 4449 8380 516 99-1506-505 C T S 4450 8381 517 99-15072-64 C T A 4451 8382 518 99-15087-77 C T A 4452 8383 519 99-15098-367 C T A 4453 8384 520 99-151-94 A G A 4454 8385 521 99-15100-363 C T A 4455 8386 522 99-15101-154 G C S 4456 8387 523 99-15106-451 C T A 4457 8388 524 99-15107-228 A G S 4458 8389 525 99-15112-358 C T A 4459 8390 526 99-15118-69 A G S 4460 8391 527 99-15123-180 C T A 4461 8392 528 99-15128-349 C T A 4462 8393 529 99-15129-279 C T A 4463 8394 530 99-15135-231 A G S 4464 8395 531 99-15137-386 A G S 4465 8396 532 99-1515-402 A G A 4466 8397 533 99-15160-270 A G S 4467 8398 534 99-15164-67 C T A 4468 8399 535 99-15193-143 G T A 4469 8400 536 99-15195-377 A C S 4470 8401 537 99-15199-179 C T A 4471 8402 538 99-1520-143 C T S 4472 8403 539 99-15200-196 A G S 4473 8404 540 99-15202-357 G C S 4474 8405 541 99-1521-457 G C S 4475 8406 542 99-1525-102 C T A 4476 8407 543 99-15290-343 G T A 4477 8408 544 99-15296-326 G C S 4478 8409 545 99-15302-371 G T A 4479 8410 546 99-15307-251 C T A 4480 8411 547 99-15310-385 A G S 4481 8412 548 99-15325-95 A G A 4482 8413 549 99-15328-328 C T A 4483 8414 550 99-1533-471 G T A 4484 8415 551 99-15330-301 G C S 4485 8416 552 99-15335-313 A G S 4486 8417 553 99-15339-378 C T A 4487 8418 554 99-15345-376 G T A 4488 8419 555 99-1535-241 C T S 4489 8420 556 99-1537-243 G T A 4490 8421 557 99-15374-99 A G S 4491 8422 558 99-15377-206 A G S 4492 8423 559 99-15382-388 G T A 4493 8424 560 99-15393-177 A T S 4494 8425 561 99-15406-220 A C S 4495 8426 562 99-15425-132 C T A 4496 8427 563 99-15441-337 A C S 4497 8428 564 99-15446-339 A G S 4498 8429 565 99-15457-171 A C S 4499 8430 566 99-15458-308 C T A 4500 8431 567 99-15473-339 A C S 4501 8432 568 99-15486-309 A C S 4502 8433 569 99-15489-305 G C S 4503 8434 570 99-1549-124 A G A 4504 8435 571 99-15490-398 A C S 4505 8436 572 99-15493-197 A G S 4506 8437 573 99-15500-77 A G S 4507 8438 574 99-15502-250 G C S 4508 8439 575 99-15503-85 G T A 4509 8440 576 99-15507-248 C T A 4510 8441 577 99-15508-259 C T A 4511 8442 578 99-15511-278 A G S 4512 8443 579 99-15516-155 A G S 4513 8444 580 99-15524-224 C T A 4514 8445 581 99-15526-324 A T S 4515 8446 582 99-15527-154 A G S 4516 8447 583 99-15528-333 A G S 4517 8448 584 99-1553-544 A C S 4518 8449 585 99-15543-55 C T A 4519 8450 586 99-15545-282 A G S 4520 8451 587 99-15557-50 A G S 4521 8452 588 99-1557-251 C T S 4522 8453 589 99-15574-261 G T A 4523 8454 590 99-15575-278 G T A 4524 8455 591 99-1558-26 C T S 4525 8456 592 99-15595-41 A G A 4526 8457 593 99-15596-64 C T S 4527 8458 594 99-15599-252 A G S 4528 8459 595 99-15605-221 A G S 4529 8460 596 99-15606-326 C T A 4530 8461 597 99-15625-299 G C S 4531 8462 598 99-15627-324 A C S 4532 8463 599 99-15636-159 A G S 4533 8464 600 99-15638-65 C T A 4534 8465 601 99-15648-83 A G S 4535 8466 602 99-15659-332 G C S 4536 8467 603 99-1568-240 A T S 4537 8468 604 99-15705-110 C T S 4538 8469 605 99-15717-120 C T A 4539 8470 606 99-15718-234 C T A 4540 8471 607 99-1572-440 C T S 4541 8472 608 99-15728-334 A T S 4542 8473 609 99-15739-113 C T A 4543 8474 610 99-15744-344 C T A 4544 8475 611 99-15747-185 C T A 4545 8476 612 99-15748-360 A C S 4546 8477 613 99-15756-54 C T A 4547 8478 614 99-15758-119 C T A 4548 8479 615 99-15762-455 A G S 4549 8480 616 99-1577-105 A G A 4550 8481 617 99-15774-268 A G S 4551 8482 618 99-15776-158 C T A 4552 8483 619 99-1578-496 C T S 4553 8484 620 99-15798-86 C T A 4554 8485 621 99-15803-52 A G S 4555 8486 622 99-15805-327 A G S 4556 8487 623 99-1582430 C T S 4557 8488 624 99-15826-407 G T A 4558 8489 625 99-15830-282 A G S 4559 8490 626 99-1585-373 C T S 4560 8491 627 99-1587-281 A G A 4561 8492 628 99-15891-215 A G S 4562 8493 629 99-15910-116 A C S 4563 8494 630 99-15916-270 A G S 4564 8495 631 99-15925-331 C T A 4565 8496 632 99-15947-109 C T A 4566 8497 633 99-15965-67 A G S 4567 8498 634 99-15966-87 C T A 4568 8499 635 99-15968-59 A G S 4569 8500 636 99-1597-162 A G A 4570 8501 637 99-15970-56 A G S 4571 8502 638 99-15973-73 C T A 4572 8503 639 99-15981-298 A G S 4573 8504 640 99-15985-354 A G S 4574 8505 641 99-15992-145 A C S 4575 8506 642 99-15996-361 G T A 4576 8507 643 99-16003-91 G C S 4577 8508 644 99-16005-314 C T A 4578 8509 645 99-1601-402 A T S 4579 8510 646 99-1602-200 G C S 4580 8511 647 99-16022-325 A G S 4581 8512 648 99-16023-160 G C S 4582 8513 649 99-16030-317 A G S 4583 8514 650 99-1605-112 A G A 4584 8515 651 99-1607-373 A G A 4585 8516 652 99-1611-382 A G A 4586 8517 653 99-16121-51 C T A 4587 8518 654 99-16128-88 C T A 4588 8519 655 99-16129-69 C T A 4589 8520 656 99-16139-79 A G S 4590 8521 657 99-16140-324 A G S 4591 8522 658 99-1615-118 A G A 4592 8523 659 99-16166-331 A G S 4593 8524 660 99-16167-130 G C S 4594 8525 661 99-16188-76 A G S 4595 8526 662 99-16192-217 A C S 4596 8527 663 99-16198-203 A T S 4597 8528 664 99-16202-240 A G S 4598 8529 665 99-16205-255 A G S 4599 8530 666 99-16210-217 A T S 4600 8531 667 99-1622-158 G C S 4601 8532 668 99-16221-161 G C S 4602 8533 669 99-16227-270 A G S 4603 8534 670 99-1623-145 C T S 4604 8535 671 99-16247-447 A G S 4605 8536 672 99-16254-304 A G S 4606 8537 673 99-16260-277 A G S 4607 8538 674 99-16262-232 C T A 4608 8539 675 99-16265-88 G C S 4609 8540 676 99-16279-409 G T A 4610 8541 677 99-16308-315 A G S 4611 8542 678 99-16346-433 C T A 4612 8543 679 99-16366-425 A T S 4613 8544 680 99-1637-345 C T S 4614 8545 681 99-16375-469 C T A 4615 8546 682 99-1638-571 C T S 4616 8547 683 99-16386-484 G C S 4617 8548 684 99-16396-174 C T A 4618 8549 685 99-16399-135 A C S 4619 8550 686 99-16403-273 A G S 4620 8551 687 99-16407-260 A C S 4621 8552 688 99-16409-58 A T S 4622 8553 689 99-16414-297 A C S 4623 8554 690 99-16445-444 C T A 4624 8555 691 99-16466-419 A C S 4625 8556 692 99-16474-299 G T A 4626 8557 693 99-16500-380 A T S 4627 8558 694 99-16505-368 G C S 4628 8559 695 99-16526-375 A C S 4629 8560 696 99-16528-194 C T A 4630 8561 697 99-16531-251 A G S 4631 8562 698 99-16535-344 C T A 4632 8563 699 99-16559-90 A G S 4633 8564 700 99-16562-182 A G S 4634 8565 701 99-16563-263 G C S 4635 8566 702 99-16564-118 A G S 4636 8567 703 99-16568-283 G T A 4637 8568 704 99-16569-65 A G S 4638 8569 705 99-1658-474 C T S 4639 8570 706 99-16611-318 A C S 4640 8571 707 99-1664-289 C T S 4641 8572 708 99-16655-135 C T S 4642 8573 709 99-16657-361 A G S 4643 8574 710 99-16677-346 C T S 4644 8575 711 99-16683-465 A C S 4645 8576 712 99-16692-343 G T A 4646 8577 713 99-16697-149 A C S 4647 8578 714 99-16708-273 A G A 4648 8579 715 99-16740-391 G T A 4649 8580 716 99-16768-54 A T S 4650 8581 717 99-16801-374 C T S 4651 8582 718 99-16827-356 A G A 4652 8583 719 99-16838-212 C T S 4653 8584 720 99-16841-120 A T S 4654 8585 721 99-16842-362 A G A 4655 8586 722 99-16845-234 A G A 4656 8587 723 99-16847-405 A G A 4657 8588 724 99-16855-84 G C S 4658 8589 725 99-16867-193 C T S 4659 8590 726 99-16873-407 G C S 4660 8591 727 99-16886-198 G T A 4661 8592 728 99-16891-264 C j A 4662 8593 729 99-16894-85 C T A 4663 8594 730 99-16895-56 A C S 4664 8595 731 99-16903-53 A G S 4665 8596 732 99-16905-281 C T A 4666 8597 733 99-16906-114 A G S 4667 8598 734 99-16913-94 A G S 4668 8599 735 99-16914-412 A G S 4669 8600 736 99-16929-479 A C S 4670 8601 737 99-16930-306 C T A 4671 8602 738 99-16933-33 A G S 4672 8603 739 99-16946-157 A C S 4673 8604 740 99-16948-390 A T S 4674 8605 741 99-16952-248 A C S 4675 8606 742 99-16975-253 G C S 4676 8607 743 99-16979-274 A G S 4677 8608 744 99-16990-155 C T A 4678 8609 745 99-16994-63 A G S 4679 8610 746 99-17001-311 C T A 4680 8611 747 99-17008-420 C T S 4681 8612 748 99-1701-39 A G A 4682 8613 749 99-17013-217 G T A 4683 8614 750 99-17016-258 A G A 4684 8615 751 99-17028-56 A G A 4685 8616 752 99-17045-267 A G A 4686 8617 753 99-17048-207 A C S 4687 8618 754 99-17052-71 A G A 4688 8619 755 99-17062-102 A G S 4689 8620 756 99-17065-230 A G S 4690 8621 757 99-17084-191 A G S 4691 8622 758 99-1709-597 G C S 4692 8623 759 99-17094-132 C T S 4693 8624 760 99-17095-424 A G S 4694 8625 761 99-1710-249 G C S 4695 8626 762 99-17103-142 C T A 4696 8627 763 99-17105-147 A G S 4697 8628 764 99-17112-191 C T A 4698 8629 765 99-17122-255 G C S 4699 8630 766 99-17123-320 C T A 4700 8631 767 99-17133-327 A T S 4701 8632 768 99-17134-82 C T A 4702 8633 769 99-17136-384 A G S 4703 8634 770 99-17154-256 A G S 4704 8635 771 99-17157-33 A G S 4705 8636 772 99-17159-280 A G S 4706 8637 773 99-17164-252 G C S 4707 8638 774 99-17165-359 A G S 4708 8639 775 99-17169-485 A T S 4709 8640 776 99-17176-315 A G S 4710 8641 777 99-17180-309 A G S 4711 8642 778 99-17204-105 A G S 4712 8643 779 99-17205-68 C T A 4713 8644 780 99-17213-128 C T A 4714 8645 781 99-1723-101 A G A 4715 8646 782 99-17253-394 G T A 4716 8647 783 99-17262-65 G T A 4717 8648 784 99-17274-353 A G A 4718 8649 785 99-17282-138 A T S 4719 8650 786 99-17306-27 C T A 4720 8651 787 99-17315-86 A T S 4721 8652 788 99-17343-305 A G S 4722 8653 789 99-17347-160 C T A 4723 8654 790 99-17351-259 G T A 4724 8655 791 99-17352-284 C T A 4725 8656 792 99-17357-244 C T A 4726 8657 793 99-17363-245 A G S 4727 8658 794 99-17365-188 A G S 4728 8659 795 99-17375-363 G C S 4729 8660 796 99-1738-72 A G A 4730 8661 797 99-17389-164 G T A 4731 8662 798 99-1739-135 A C S 4732 8663 799 99-17409-293 C T S 4733 8664 800 99-17412-296 C T A 4734 8665 801 99-17416-310 A G S 4735 8666 802 99-17418-41 A T S 4736 8667 803 99-17420-380 A G S 4737 8668 804 99-17428-129 A G S 4738 8669 805 99-17450-352 A G S 4739 8670 806 99-17464-376 C T A 4740 8671 807 99-17476-141 A C S 4741 8672 808 99-17481-171 C T A 4742 8673 809 99-17483-282 G T A 4743 8674 810 99-17490-199 A G S 4744 8675 811 99-17491-362 C T A 4745 8676 812 99-17495-100 A C S 4746 8677 813 99-17496-301 A C S 4747 8678 814 99-17498-312 C T A 4748 8679 815 99-17499-62 A G S 4749 8680 816 99-17520-31 C T A 4750 8681 817 99-17522-423 A G S 4751 8682 818 99-17523-116 A C S 4752 8683 819 99-17529-210 A G S 4753 8684 820 99-17557-358 C T A 4754 8685 821 99-17563-102 G C S 4755 8686 822 99-17588-501 A G S 4756 8687 823 99-17610-44 A G S 4757 8688 824 99-17629-89 G T A 4758 8689 825 99-1764-65 G C S 4759 8690 826 99-17647-79 A G S 4760 8691 827 99-1765-171 C T S 4761 8692 828 99-17656-239 A G S 4762 8693 829 99-17658-167 A G S 4763 8694 830 99-17662-126 C T A 4764 8695 831 99-17663-29 A G S 4765 8696 832 99-17677-251 A G A 4766 8697 833 99-17680-451 C T S 4767 8698 834 99-17683-286 A G A 4768 8699 835 99-17687-373 C T S 4769 8700 836 99-17700-191 G C S 4770 8701 837 99-17702-57 G T A 4771 8702 838 99-17718-259 C T S 4772 8703 839 99-17720-224 G C S 4773 8704 840 99-17728-310 A C S 4774 8705 841 99-1773-343 C T S 4775 8706 842 99-17740-227 G C S 4776 8707 843 99-1775-187 A G A 4777 8708 844 99-17758-292 A G A 4778 8709 845 99-17762-327 A G A 4779 8710 846 99-17773-392 C T S 4780 8711 847 99-17774-276 C T S 4781 8712 848 99-17775-286 C T S 4782 8713 849 99-17776-114 A T S 4783 8714 850 99-17779-117 A C S 4784 8715 851 99-17792-144 C T S 4785 8716 852 99-17798-345 C T S 4786 8717 853 99-17802-338 A G A 4787 8718 854 99-17808-398 G C S 4788 8719 855 99-1781-129 A G A 4789 8720 856 99-17810-366 C T S 4790 8721 857 99-17816-377 G C S 4791 8722 858 99-17820-316 A G A 4792 8723 859 99-17821-109 A G S 4793 8724 860 99-17827-106 A C S 4794 8725 861 99-17829-412 G C S 4795 8726 862 99-17833-108 G T A 4796 8727 863 99-17845-286 A G A 4797 8728 864 99-17854-229 C T S 4798 8729 865 99-17856-308 G C S 4799 8730 866 99-17857-251 A G A 4800 8731 867 99-17863-257 A G A 4801 8732 868 99-17864-202 G T A 4802 8733 869 99-17866-124 C T S 4803 8734 870 99-17889-148 G C S 4804 8735 871 99-17890-58 C T S 4805 8736 872 99-17899-140 A G A 4806 8737 873 99-17913-222 G T A 4807 8738 874 99-17920-382 A G A 4808 8739 875 99-1793-225 A T S 4809 8740 876 99-17938-131 A G A 4810 8741 877 99-17945-63 A T S 4811 8742 878 99-17946-69 A C S 4812 8743 879 99-17952-370 C T S 4813 8744 880 99-1796-184 A T S 4814 8745 881 99-17971-78 A G A 4815 8746 882 99-17976-132 G C S 4816 8747 883 99-17989-85 A G A 4817 8748 884 99-17991-412 A T S 4818 8749 885 99-17992-404 A G A 4819 8750 886 99-18004-125 C T S 4820 8751 887 99-18007-159 C T S 4821 8752 888 99-18030-54 A T S 4822 8753 889 99-18038-384 G C S 4823 8754 890 99-18046-65 A T S 4824 8755 891 99-18053-328 A G S 4825 8756 892 99-18054-392 A C S 4826 8757 893 99-18056-354 A G S 4827 8758 894 99-18057-55 A C S 4828 8759 895 99-18060-203 G T A 4829 8760 896 99-18062-187 A G A 4830 8761 897 99-18069-282 C T A 4831 8762 898 99-18079-46 G C S 4832 8763 899 99-1808-291 A T S 4833 8764 900 99-18080-378 G T A 4834 8765 901 99-18085-94 A G S 4835 8766 902 99-18086-434 A G S 4836 8767 903 99-18087-152 C T A 4837 8768 904 99-18091-47 G C S 4838 8769 905 99-18096-198 C T A 4839 8770 906 99-18109-159 C T A 4840 8771 907 99-1813-310 C T S 4841 8772 908 99-18130-258 A G S 4842 8773 909 99-1814-245 A G A 4843 8774 910 99-18171-95 G T A 4844 8775 911 99-18172-284 A G S 4845 8776 912 99-18179-185 G T A 4846 8777 913 99-18198-203 C T A 4847 8778 914 99-18201-23 A G S 4848 8779 915 99-18206-76 A G S 4849 8780 916 99-18210-30 G C S 4850 8781 917 99-18213-185 A G S 4851 8782 918 99-18214-86 A C S 4852 8783 919 99-18221-207 C T A 4853 8784 920 99-1823-157 A G A 4854 8785 921 99-1824-226 A G A 4855 8786 922 99-18242-369 A G S 4856 8787 923 99-18253-407 C T S 4857 8788 924 99-18255-259 A T S 4858 8789 925 99-18258-45 G C S 4859 8790 926 99-18268-460 A G A 4860 8791 927 99-18272-287 G C S 4861 8792 928 99-18276-390 A G S 4862 8793 929 99-18288-205 A G A 4863 8794 930 99-18289-36 C T S 4864 8795 931 99-18303-79 C T S 4865 8796 932 99-18306-377 A G A 4866 8797 933 99-18307-371 A C S 4867 8798 934 99-18310-262 C T S 4868 8799 935 99-18312-58 C T S 4869 8800 936 99-18341-95 G T A 4870 8801 937 99-18344-284 A G A 4871 8802 938 99-18345-107 C T S 4872 8803 939 99-18371-433 A T S 4873 8804 940 99-18373-27 A G A 4874 8805 941 99-18375-237 A G A 4875 8806 942 99-18379-485 C T S 4876 8807 943 99-18386-177 A T S 4877 8808 944 99-18394-132 G T A 4878 8809 945 99-18402-255 C T S 4879 8810 946 99-18406-155 A G A 4880 8811 947 99-18414-204 A C S 4881 8812 948 99-18418-127 G C S 4882 8813 949 99-1842-78 C T S 4883 8814 950 99-18423-336 A G A 4884 8815 951 99-18427-314 C T S 4885 8816 952 99-18438-398 C T S 4886 8817 953 99-18442-283 C T S 4887 8818 954 99-18444-185 C T S 4888 8819 955 99-18458-191 A G A 4889 8820 956 99-18470-119 A T S 4890 8821 957 99-18478-101 A G A 4891 8822 958 99-18486-49 A G A 4892 8823 959 99-18487-236 C T S 4893 8824 960 99-18488-273 A G A 4894 8825 961 99-1849-421 C T S 4895 8826 962 99-18536-290 G T A 4896 8827 963 99-18542-232 A C S 4897 8828 964 99-18551-389 G C S 4898 8829 965 99-18561-371 A C S 4899 8830 966 99-18573-363 G T A 4900 8831 967 99-18582-422 A G A 4901 8832 968 99-18588-175 A C S 4902 8833 969 99-18596-83 A G A 4903 8834 970 99-18597-415 C T S 4904 8835 971 99-18599-347 A G A 4905 8836 972 99-1860-281 A G A 4906 8837 973 99-18602-241 A G A 4907 8838 974 99-18606-324 C T S 4908 8839 975 99-1861-191 G C S 4909 8840 976 99-18612-184 C T S 4910 8841 977 99-18618-455 C T S 4911 8842 978 99-18620-125 C T S 4912 8843 979 99-18637-281 C T A 4913 8844 980 99-18638-164 C T A 4914 8845 981 99-18640-458 C T A 4915 8846 982 99-18648-71 C T A 4916 8847 983 99-18666-483 G T A 4917 8848 984 99-18667-392 G C S 4918 8849 985 99-18669-223 G C S 4919 8850 986 99-18715-172 A G A 4920 8851 987 99-18719-225 C T S 4921 8852 988 99-18720-235 C T S 4922 8853 989 99-18721-442 A G A 4923 8854 990 99-18724-409 C T S 4924 8855 991 99-18729-377 A T S 4925 8856 992 99-1873-193 C T A 4926 8857 993 99-18744-170 A G A 4927 8858 994 99-18745-423 A G A 4928 8859 995 99-18747-72 C T S 4929 8860 996 99-18751-217 G T A 4930 8861 997 99-18755-267 C T S 4931 8862 998 99-18774-69 G T A 4932 8863 999 99-18775-161 G T A 4933 8864 1000 99-18777-130 C T S 4934 8865 1001 99-18802-308 G C S 4935 8866 1002 99-18808-155 C T S 4936 8867 1003 99-18814-275 G C S 4937 8868 1004 99-1882-289 C T A 4938 8869 1005 99-18822-368 C T S 4939 8870 1006 99-18826-378 C T S 4940 8871 1007 99-18827-92 A G A 4941 8872 1008 99-1883-121 G T A 4942 8873 1009 99-18847-263 C T S 4943 8874 1010 99-18853-64 G T A 4944 8875 1011 99-18855-173 A G A 4945 8876 1012 99-18860-308 C T S 4946 8877 1013 99-18861-23 C T S 4947 8878 1014 99-1888-162 C T S 4948 8879 1015 99-1890-125 C T A 4949 8880 1016 99-1895-67 A C S 4950 8881 1017 99-18974-99 A G A 4951 8882 1018 99-18976-135 A T S 4952 8883 1019 99-18982-345 C T S 4953 8884 1020 99-18986-248 G C S 4954 8885 1021 99-18987-191 A G A 4955 8886 1022 99-18995-300 C T S 4956 8887 1023 99-18996-388 A G A 4957 8888 1024 99-19008-237 C T S 4958 8889 1025 99-19013-384 C T S 4959 8890 1026 99-19016-51 A G A 4960 8891 1027 99-1909-387 G T A 4961 8892 1028 99-1910-94 C T S 4962 8893 1029 99-1916-91 G T A 4963 8894 1030 99-1917-434 A T S 4964 8895 1031 99-19253-102 A G A 4965 8896 1032 99-19256-149 C T S 4966 8897 1033 99-1934-272 A G A 4967 8898 1034 99-1936-289 C T S 4968 8899 1035 99-1944-379 C T S 4969 8900 1036 99-1947-205 A G S 4970 8901 1037 99-1948-49 G C S 4971 8902 1038 99-1953-287 A G A 4972 8903 1039 99-1955-443 A G A 4973 8904 1040 99-1960-424 A T S 4974 8905 1041 99-1964-53 C T S 4975 8906 1042 99-1977-440 A G S 4976 8907 1043 99-1997-139 G T A 4977 8908 1044 99-19999-92 C T S 4978 8909 1045 99-2000-240 G T A 4979 8910 1046 99-20000-252 A G A 4980 8911 1047 99-2001-177 A G S 4981 8912 1048 99-20011-229 C T S 4982 8913 1049 99-20018-244 G C S 4983 8914 1050 99-20023-386 A T S 4984 8915 1051 99-2003-509 G C S 4985 8916 1052 99-20032-90 G T A 4986 8917 1053 99-20033-186 G T A 4987 8918 1054 99-20035-283 A C S 4988 8919 1055 99-2004-35 C T S 4989 8920 1056 99-2005-466 G C S 4990 8921 1057 99-20057-166 C T S 4991 8922 1058 99-20061-56 C T S 4992 8923 1059 99-20062-181 A G A 4993 8924 1060 99-2007-278 C T S 4994 8925 1061 99-20074-154 A C S 4995 8926 1062 99-20090-81 A C S 4996 8927 1063 99-2010-363 C T A 4997 8928 1064 99-20110-65 G T A 4998 8929 1065 99-2012-243 A G A 4999 8930 1066 99-20154-451 A T S 5000 8931 1067 99-20156-212 A G A 5001 8932 1068 99-20198-54 C T S 5002 8933 1069 99-2020-281 C T A 5003 8934 1070 99-20208-176 A G A 5004 8935 1071 99-2022-200 A C S 5005 8936 1072 99-2024-132 A G A 5006 8937 1073 99-2025-234 C T A 5007 8938 1074 99-20250-362 A T S 5008 8939 1075 99-2027-296 A G S 5009 8940 1076 99-20294-274 C T S 5010 8941 1077 99-20303-127 C T S 5011 8942 1078 99-20313-311 A G A 5012 8943 1079 99-20320-321 C T S 5013 8944 1080 99-20326-130 A G A 5014 8945 1081 99-20332-432 A G A 5015 8946 1082 99-20335-48 C T S 5016 8947 1083 99-20340-161 A G A 5017 8948 1084 99-20348-403 A G A 5018 8949 1085 99-2035-323 C T S 5019 8950 1086 99-20353-229 A G A 5020 8951 1087 99-20357-359 A T S 5021 8952 1088 99-2036-168 A T S 5022 8953 1089 99-2037-470 C T S 5023 8954 1090 99-20385-215 C T S 5024 8955 1091 99-2041-141 A G A 5025 8956 1092 99-2042-439 G C S 5026 8957 1093 99-20420-274 C T S 5027 8958 1094 99-20423-430 C T S 5028 8959 1095 99-20424-330 C T S 5029 8960 1096 99-20428-271 C T S 5030 8961 1097 99-2043-220 A T S 5031 8962 1098 99-2046-275 A G A 5032 8963 1099 99-20469-213 C T S 5033 8964 1100 99-2048-267 G C S 5034 8965 1101 99-20480-233 C T S 5035 8966 1102 99-20481-131 G C S 5036 8967 1103 99-20485-269 A G A 5037 8968 1104 99-20493-238 G T A 5038 8969 1105 99-20499-364 A T S 5039 8970 1106 99-20504-90 A G A 5040 8971 1107 99-20508-456 C T S 5041 8972 1108 99-2051-360 A C S 5042 8973 1109 99-20511-221 C T S 5043 8974 1110 99-20514-71 A G A 5044 8975 1111 99-20518-456 A G A 5045 8976 1112 99-2052-376 G T A 5046 8977 1113 99-20527-220 A T S 5047 8978 1114 99-2053-386 A G A 5048 8979 1115 99-20531-285 A C S 5049 8980 1116 99-2054-93 A G S 5050 8981 1117 99-20542-248 A G A 5051 8982 1118 99-20549-141 A G A 5052 8983 1119 99-2055-236 A G A 5053 8984 1120 99-20552-37 C T S 5054 8985 1121 99-2056-474 C T S 5055 8986 1122 99-20561-126 G T A 5056 8987 1123 99-20565-190 C T S 5057 8988 1124 99-20566-376 A G A 5058 8989 1125 99-20567-268 C T S 5059 8990 1126 99-20568-284 A C S 5060 8991 1127 99-2058-168 G T A 5061 8992 1128 99-20581-125 A T S 5062 8993 1129 99-20594-103 G C S 5063 8994 1130 99-2060-322 A T S 5064 8995 1131 99-2061-257 A C S 5065 8996 1132 99-20616-287 C T S 5066 8997 1133 99-20623-354 C T S 5067 8998 1134 99-2063-451 A G A 5068 8999 1135 99-20639-257 C T S 5069 9000 1136 99-20642-382 A G A 5070 9001 1137 99-20651-108 A G A 5071 9002 1138 99-20656-171 C T S 5072 9003 1139 99-20659-289 C T S 5073 9004 1140 99-20675-407 G C S 5074 9005 1141 99-20677-289 C T S 5075 9006 1142 99-20683-98 A C S 5076 9007 1143 99-20688-310 A G A 5077 9008 1144 99-20723-206 C T S 5078 9009 1145 99-20726-494 A G A 5079 9010 1146 99-20732-413 G C S 5080 9011 1147 99-20738-89 G C S 5081 9012 1148 99-20739-335 A G A 5082 9013 1149 99-2074-273 A C S 5083 9014 1150 99-20746-369 A G A 5084 9015 1151 99-20747-322 A G A 5085 9016 1152 99-20766-117 A G A 5086 9017 1153 99-20768-469 C T S 5087 9018 1154 99-2077-510 G C S 5088 9019 1155 99-20771-171 A G A 5089 9020 1156 99-2078-348 A G A 5090 9021 1157 99-20797-262 C T S 5091 9022 1158 99-20798-87 C T S 5092 9023 1159 99-2080-33 A G A 5093 9024 1160 99-20802-358 C T S 5094 9025 1161 99-20814-222 C T S 5095 9026 1162 99-2082-284 A G A 5096 9027 1163 99-20823-49 C T S 5097 9028 1164 99-20828-131 C T S 5098 9029 1165 99-20830-449 A G A 5099 9030 1166 99-2084-504 A G A 5100 9031 1167 99-2085-172 C T S 5101 9032 1168 99-20850-374 C T S 5102 9033 1169 99-20853-29 A G A 5103 9034 1170 99-20856-158 C T S 5104 9035 1171 99-20867-393 A G A 5105 9036 1172 99-20872-325 A C S 5106 9037 1173 99-20883-234 C T S 5107 9038 1174 99-20887-420 C T S 5108 9039 1175 99-2089-84 A G A 5109 9040 1176 99-20895-36 A G S 5110 9041 1177 99-2092-323 A C S 5111 9042 1178 99-20928-66 G C S 5112 9043 1179 99-2093-278 C T S 5113 9044 1180 99-20938-256 G T A 5114 9045 1181 99-2094-129 A G A 5115 9046 1182 99-20950-251 G C S 5116 9047 1183 99-2098-102 G T A 5117 9048 1184 99-21012-277 A C S 5118 9049 1185 99-21021-273 C T S 5119 9050 1186 99-2103-270 G C S 5120 9051 1187 99-21035-279 C T S 5121 9052 1188 99-21064-278 C T S 5122 9053 1189 99-21070-272 A G A 5123 9054 1190 99-21079-169 G T A 5124 9055 1191 99-21084-496 C T S 5125 9056 1192 99-2109-276 G T A 5126 9057 1193 99-211-291 A G S 5127 9058 1194 99-21141-314 A G A 5128 9059 1195 99-21148-269 A G A 5129 9060 1196 99-21149-129 A G A 5130 9061 1197 99-21167-159 C T S 5131 9062 1198 99-2117-107 C T S 5132 9063 1199 99-21221-96 A T S 5133 9064 1200 99-2126-79 A T S 5134 9065 1201 99-21370-87 C T S 5135 9066 1202 99-2170-188 G C S 5136 9067 1203 99-2172-314 A G S 5137 9068 1204 99-2173-289 C T S 5138 9069 1205 99-2179-303 G T A 5139 9070 1206 99-2193-225 A G A 5140 9071 1207 99-22011-342 C T S 5141 9072 1208 99-22015-219 A G A 5142 9073 1209 99-22022-145 A G A 5143 9074 1210 99-22027-410 G C S 5144 9075 1211 99-22036-314 A T S 5145 9076 1212 99-22038-381 G C S 5146 9077 1213 99-22044-431 A G A 5147 9078 1214 99-22048-259 A G A 5148 9079 1215 99-22051-261 C T S 5149 9080 1216 99-22066-139 A G A 5150 9081 1217 99-22072-80 C T S 5151 9082 1218 99-22073-381 G C S 5152 9083 1219 99-22078-350 A G A 5153 9084 1220 99-22087-150 C T S 5154 9085 1221 99-2209-111 A G A 5155 9086 1222 99-22091-289 G T A 5156 9087 1223 99-22096-276 C T S 5157 9088 1224 99-22100-265 C T S 5158 9089 1225 99-22102-238 C T S 5159 9090 1226 99-22122-54 A G A 5160 9091 1227 99-22125-126 C T S 5161 9092 1228 99-2214-148 A C S 5162 9093 1229 99-22147-359 C T S 5163 9094 1230 99-22160-331 A G A 5164 9095 1231 99-22167-79 C T S 5165 9096 1232 99-22172-304 A T S 5166 9097 1233 99-2218-219 A G A 5167 9098 1234 99-22189-248 C T S 5168 9099 1235 99-2219-245 C T S 5169 9100 1236 99-22191-339 C T S 5170 9101 1237 99-22192-383 C T S 5171 9102 1238 99-222-109 C T S 5172 9103 1239 99-2220-300 A G A 5173 9104 1240 99-22209-304 A C S 5174 9105 1241 99-22215-391 A G A 5175 9106 1242 99-22217-423 G C S 5176 9107 1243 99-2222-459 C T S 5177 9108 1244 99-22227-275 A T S 5178 9109 1245 99-22255-384 A G A 5179 9110 1246 99-22262-331 C T S 5180 9111 1247 99-22265-294 A C S 5181 9112 1248 99-22266-474 C T S 5182 9113 1249 99-2228-301 A G A 5183 9114 1250 99-2229-240 G T A 5184 9115 1251 99-22333-237 C T S 5185 9116 1252 99-22336-316 C T S 5186 9117 1253 99-22337-199 A C S 5187 9118 1254 99-2235-499 G C S 5188 9119 1255 99-22356-370 A C S 5189 9120 1256 99-22357-186 C T S 5190 9121 1257 99-2240-281 C T S 5191 9122 1258 99-22409-141 C T S 5192 9123 1259 99-2242-206 C T S 5193 9124 1260 99-2244-83 A G A 5194 9125 1261 99-22442-147 G T A 5195 9126 1262 99-22449-216 G C S 5196 9127 1263 99-22453-370 A T S 5197 9128 1264 99-22456-55 A C S 5198 9129 1265 99-2246-340 A G A 5199 9130 1266 99-2248-76 C T S 5200 9131 1267 99-22490-246 A G A 5201 9132 1268 99-22491-79 G T A 5202 9133 1269 99-2250-236 C T S 5203 9134 1270 99-22503-146 C T S 5204 9135 1271 99-22506-395 C T S 5205 9136 1272 99-22513-90 A G A 5206 9137 1273 99-22520-413 G C S 5207 9138 1274 99-22546-125 C T S 5208 9139 1275 99-22565-114 A G A 5209 9140 1276 99-22571-136 C T S 5210 9141 1277 99-22573-321 A G A 5211 9142 1278 99-22578-78 C T S 5212 9143 1279 99-22580-72 A T S 5213 9144 1280 99-22585-462 G C S 5214 9145 1281 99-22586-39 A G S 5215 9146 1282 99-22604-208 G T A 5216 9147 1283 99-22610-343 A G A 5217 9148 1284 99-22615-392 C T S 5218 9149 1285 99-22617-378 C T S 5219 9150 1286 99-22620-404 C T S 5220 9151 1287 99-22628-292 A G A 5221 9152 1288 99-22629-124 C T S 5222 9153 1289 99-22632-237 G C S 5223 9154 1290 99-22646-233 A G A 5224 9155 1291 99-22648-57 C T S 5225 9156 1292 99-22650-64 A C S 5226 9157 1293 99-22652-343 A G A 5227 9158 1294 99-22655-319 A T S 5228 9159 1295 99-22660-386 A G A 5229 9160 1296 99-22662-268 A G A 5230 9161 1297 99-22666-164 C T S 5231 9162 1298 99-22668-232 G T A 5232 9163 1299 99-22674-31 C T S 5233 9164 1300 99-22675-187 A G A 5234 9165 1301 99-22680-130 C T S 5235 9166 1302 99-22683-107 A G A 5236 9167 1303 99-2269-179 A G A 5237 9168 1304 99-22700-358 A G A 5238 9169 1305 99-22701-307 C T S 5239 9170 1306 99-2271-403 A G A 5240 9171 1307 99-22712-242 A G A 5241 9172 1308 99-22718-94 A T S 5242 9173 1309 99-2272-409 G T A 5243 9174 1310 99-22728-207 A G A 5244 9175 1311 99-2273-528 C T S 5245 9176 1312 99-22733-281 G C S 5246 9177 1313 99-22741-180 A G A 5247 9178 1314 99-2275-466 C T S 5248 9179 1315 99-2276-331 C T S 5249 9180 1316 99-22771-150 A G A 5250 9181 1317 99-22775-365 C T S 5251 9182 1318 99-2278-276 A G A 5252 9183 1319 99-22785-431 A T S 5253 9184 1320 99-22843-342 G T A 5254 9185 1321 99-22844-211 A G A 5255 9186 1322 99-22857-88 C T S 5256 9187 1323 99-22868-425 A C S 5257 9188 1324 99-22872-431 C T S 5258 9189 1325 99-2288-144 C T S 5259 9190 1326 99-22917-145 G T A 5260 9191 1327 99-22937-395 C T S 5261 9192 1328 99-22948-262 C T S 5262 9193 1329 99-22954-306 A C S 5263 9194 1330 99-22957-409 A G A 5264 9195 1331 99-22959-239 A G A 5265 9196 1332 99-22964-82 C T S 5266 9197 1333 99-22975-126 C T S 5267 9198 1334 99-23014-300 A G A 5268 9199 1335 99-23018-166 A G A 5269 9200 1336 99-23020-187 G T A 5270 9201 1337 99-23083-59 C T S 5271 9202 1338 99-23100-367 A G A 5272 9203 1339 99-23115-404 G C S 5273 9204 1340 99-23118-402 A G A 5274 9205 1341 99-2312-358 C T S 5275 9206 1342 99-23123-250 A G A 5276 9207 1343 99-23127-314 G C S 5277 9208 1344 99-23132-192 A G A 5278 9209 1345 99-23134-89 A G A 5279 9210 1346 99-2315-213 A G A 5280 9211 1347 99-23150-262 A C S 5281 9212 1348 99-2320-292 C T S 5282 9213 1349 99-23201-345 C T S 5283 9214 1350 99-23202-185 A C S 5284 9215 1351 99-23204-262 C T S 5285 9216 1352 99-23207-281 C T S 5286 9217 1353 99-2321-82 C T S 5287 9218 1354 99-23228-176 G C S 5288 9219 1355 99-2324-338 A C S 5289 9220 1356 99-23266-146 A G A 5290 9221 1357 99-23269-263 A T S 5291 9222 1358 99-2328-535 G C S 5292 9223 1359 99-23299-424 A G A 5293 9224 1360 99-23302-326 C T S 5294 9225 1361 99-2331-639 G T A 5295 9226 1362 99-23312-93 A G A 5296 9227 1363 99-23317-51 A G A 5297 9228 1364 99-23322-49 A G A 5298 9229 1365 99-23326-120 A G A 5299 9230 1366 99-23328-292 A G A 5300 9231 1367 99-23333-157 A G A 5301 9232 1368 99-23334-443 A G A 5302 9233 1369 99-23359-99 G C S 5303 9234 1370 99-23381-412 A G A 5304 9235 1371 99-23387-404 G C S 5305 9236 1372 99-23413-242 A G A 5306 9237 1373 99-23415-131 A G A 5307 9238 1374 99-23417-128 G T A 5308 9239 1375 99-23437-347 A G A 5309 9240 1376 99-23440-274 A G A 5310 9241 1377 99-23444-203 A G A 5311 9242 1378 99-2345-28 G C A 5312 9243 1379 99-23451-78 A G A 5313 9244 1380 99-23452-306 G T A 5314 9245 1381 99-23454-317 C T A 5315 9246 1382 99-23460-199 A C S 5316 9247 1383 99-23462-192 C T S 5317 9248 1384 99-23463-118 C T S 5318 9249 1385 99-23469-288 C T S 5319 9250 1386 99-2347-207 A C S 5320 9251 1387 99-23473-35 C T S 5321 9252 1388 99-2348-127 A G A 5322 9253 1389 99-23488-239 A G A 5323 9254 1390 99-23492-151 C T S 5324 9255 1391 99-23496-94 A G A 5325 9256 1392 99-2351045 A G A 5326 9257 1393 99-23528-452 C T S 5327 9258 1394 99-2356-322 A G A 5328 9259 1395 99-2362-270 A G A 5329 9260 1396 99-2364-329 G C S 5330 9261 1397 99-2367-61 A G A 5331 9262 1398 99-2368-61 A G A 5332 9263 1399 99-23687-107 C T A 5333 9264 1400 99-237-151 A G A 5334 9265 1401 99-23714-196 G C S 5335 9266 1402 99-23737-186 C T S 5336 9267 1403 99-2375-114 C T A 5337 9268 1404 99-23773-199 C T S 5338 9269 1405 99-2378-200 A G A 5339 9270 1406 99-2381-394 A G A 5340 9271 1407 99-2409-298 A G A 5341 9272 1408 99-241-341 A T S 5342 9273 1409 99-2413-368 A G A 5343 9274 1410 99-2417-177 C T S 5344 9275 1411 99-2419-285 C T S 5345 9276 1412 99-24246-247 C T A 5346 9277 1413 99-24253-437 A G S 5347 9278 1414 99-24259-466 A T S 5348 9279 1415 99-24264-380 G C S 5349 9280 1416 99-24269-417 C T A 5350 9281 1417 99-24270-207 G T A 5351 9282 1418 99-24275-107 A G S 5352 9283 1419 99-24284-213 A T S 5353 9284 1420 99-24286-231 A G S 5354 9285 1421 99-24288-121 A G S 5355 9286 1422 99-24333-37 A G S 5356 9287 1423 99-24342-311 C T A 5357 9288 1424 99-24376-24 A G S 5358 9289 1425 99-24379-319 C T S 5359 9290 1426 99-24381-217 A G S 5360 9291 1427 99-24385-210 A T S 5361 9292 1428 99-24388-391 A T S 5362 9293 1429 99-24390-27 A G S 5363 9294 1430 99-24392-61 A C S 5364 9295 1431 99-24393-108 A G S 5365 9296 1432 99-2440-246 C T S 5366 9297 1433 99-24409-383 A G S 5367 9298 1434 99-24411-420 G C S 5368 9299 1435 99-24427-321 A G S 5369 9300 1436 99-24432-284 A C S 5370 9301 1437 99-24438-402 A G S 5371 9302 1438 99-24441-431 C T A 5372 9303 1439 99-24447-448 A T S 5373 9304 1440 99-2445-79 C T S 5374 9305 1441 99-24454-257 G C S 5375 9306 1442 99-24463-206 A G S 5376 9307 1443 99-24496-171 C T A 5377 9308 1444 99-24506-396 A G S 5378 9309 1445 99-24508-45 G C S 5379 9310 1446 99-24529-330 A G S 5380 9311 1447 99-24534-317 G C S 5381 9312 1448 99-24554-324 A G A 5382 9313 1449 99-24557-406 G T A 5383 9314 1450 99-24561-360 A T S 5384 9315 1451 99-24570-260 G C S 5385 9316 1452 99-24688-312 C T A 5386 9317 1453 99-24725-138 A G S 5387 9318 1454 99-24727-360 A G S 5388 9319 1455 99-24750-293 C T A 5389 9320 1456 99-24778-221 C T A 5390 9321 1457 99-24793-390 C T A 5391 9322 1458 99-24800-565 G T S 5392 9323 1459 99-25005-154 A C S 5393 9324 1460 99-25007-131 A T S 5394 9325 1461 99-25053-114 A G S 5395 9326 1462 99-25055-44 A G S 5396 9327 1463 99-25070-78 C T A 5397 9328 1464 99-25077-124 A G S 5398 9329 1465 99-25129-166 C T A 5399 9330 1466 99-25134-296 A G S 5400 9331 1467 99-2524-98 A G A 5401 9332 1468 99-25246-170 C T A 5402 9333 1469 99-25249-151 G T A 5403 9334 1470 99-2525-142 A G S 5404 9335 1471 99-25255-288 C T A 5405 9336 1472 99-25369-121 C T S 5406 9337 1473 99-25379-389 C T S 5407 9338 1474 99-25382-226 A C S 5408 9339 1475 99-25387-220 G T A 5409 9340 1476 99-25400-379 C T S 5410 9341 1477 99-25412-354 G T A 5411 9342 1478 99-25431-269 A C S 5412 9343 1479 99-25432-119 C T S 5413 9344 1480 99-25433-351 A G S 5414 9345 1481 99-25447-272 A G S 5415 9346 1482 99-25448-348 G T A 5416 9347 1483 99-25452-83 C T A 5417 9348 1484 99-25454-349 G T A 5418 9349 1485 99-25458-103 G C S 5419 9350 1486 99-25503-333 A T S 5420 9351 1487 99-25507-373 C T A 5421 9352 1488 99-25510-390 A C S 5422 9353 1489 99-25538-423 A C S 5423 9354 1490 99-25539-86 C T A 5424 9355 1491 99-25543-390 A G S 5425 9356 1492 99-25575-303 C T A 5426 9357 1493 99-25618-196 A C S 5427 9358 1494 99-25620-360 C T A 5428 9359 1495 99-25629-262 A G S 5429 9360 1496 99-25657-314 C T A 5430 9361 1497 99-25672-97 A G S 5431 9362 1498 99-25676-211 C T A 5432 9363 1499 99-25678-307 A G S 5433 9364 1500 99-2570-218 C T S 5434 9365 1501 99-25712-418 C T A 5435 9366 1502 99-25716-393 C T A 5436 9367 1503 99-25717-252 G T A 5437 9368 1504 99-25725-80 A G S 5438 9369 1505 99-25732-152 A G S 5439 9370 1506 99-25745-36 A G S 5440 9371 1507 99-25781-275 C T A 5441 9372 1508 99-25836-106 C T S 5442 9373 1509 99-2597-34 C T S 5443 9374 1510 99-26001-224 A G S 5444 9375 1511 99-26002-93 C T A 5445 9376 1512 99-26042-310 A G A 5446 9377 1513 99-26080-152 C T S 5447 9378 1514 99-26082-48 C T S 5448 9379 1515 99-26099-119 A G A 5449 9380 1516 99-2610-121 A C S 5450 9381 1517 99-26105-273 A C S 5451 9382 1518 99-26116-191 C T A 5452 9383 1519 99-2615-83 C T S 5453 9384 1520 99-2620-227 A G A 5454 9385 1521 99-2624-407 G T A 5455 9386 1522 99-2625-70 A G A 5456 9387 1523 99-2637-28 A G A 5457 9388 1524 99-2662-407 C T S 5458 9389 1525 99-2669-233 A G A 5459 9390 1526 99-2675-121 A G A 5460 9391 1527 99-2683-388 C T S 5461 9392 1528 99-342-288 A C S 5462 9393 1529 99-370-205 A G A 5463 9394 1530 99-371-415 C T S 5464 9395 1531 99-388-405 A G A 5465 9396 1532 99-390-246 G T S 5466 9397 1533 99-393-448 A T S 5467 9398 1534 99-397-205 A G A 5468 9399 1535 99-400-102 G C S 5469 9400 1536 99-402-139 A G A 5470 9401 1537 99-404-114 C T S 5471 9402 1538 99-414-349 G C S 5472 9403 1539 99-417-241 A G A 5473 9404 1540 99-426-359 G T S 5474 9405 1541 99-429-115 A C S 5475 9406 1542 99-430-352 C T S 5476 9407 1543 99-435-41 A G A 5477 9408 1544 99-449-344 G T S 5478 9409 1545 99-4536-255 A G A 5479 9410 1546 99-4541-39 G T A 5480 9411 1547 99-4544-287 A G A 5481 9412 1548 99-4547-312 C T S 5482 9413 1549 99-4595-341 A T S 5483 9414 1550 99-4604-26 A T S 5484 9415 1551 994618-240 C T S 5485 9416 1552 99-4625-216 C T S 5486 9417 1553 99-4630-272 A G A 5487 9418 1554 99-4644-107 A T S 5488 9419 1555 99-465-443 A C S 5489 9420 1556 99-4655-145 C T S 5490 9421 1557 99-466-361 C T S 5491 9422 1558 99-4666-185 C T S 5492 9423 1559 99-4674-166 A G A 5493 9424 1560 99-4676-342 A G S 5494 9425 1561 99-4677-58 C T S 5495 9426 1562 99-4679-240 C T A 5496 9427 1563 99-4680-352 C T S 5497 9428 1564 99-4681-228 A G A 5498 9429 1565 99-4682-177 C T S 5499 9430 1566 99-4685-217 A G A 5500 9431 1567 99-4705-226 G C S 5501 9432 1568 99-4714-156 A G A 5502 9433 1569 99-472-70 C T S 5503 9434 1570 99-4725-251 A T S 5504 9435 1571 99-4756-236 C T S 5505 9436 1572 99-4761-279 A G A 5506 9437 1573 99-477-302 A G A 5507 9438 1574 99-4777-137 G C S 5508 9439 1575 99-4790-305 A G A 5509 9440 1576 99-4796-325 A C S 5510 9441 1577 99-482-130 C T S 5511 9442 1578 99-4822-291 A G A 5512 9443 1579 994823-173 G T A 5513 9444 1580 99-483-424 A C S 5514 9445 1581 99-4836-206 A G A 5515 9446 1582 99-4838-424 C T S 5516 9447 1583 99-4840-368 C T S 5517 9448 1584 99-4844-102 C T S 5518 9449 1585 99-486-243 C T S 5519 9450 1586 99-4863-240 C T S 5520 9451 1587 99-4882-351 A T S 5521 9452 1588 99-4890-255 G T A 5522 9453 1589 99-4891-509 A C S 5523 9454 1590 99-4895-158 C T S 5524 9455 1591 99-490-202 C T S 5525 9456 1592 99-4924-254 C T S 5526 9457 1593 99-4928-102 C T S 5527 9458 1594 99-4950-196 A C S 5528 9459 1595 99-4951-36 C T A 5529 9460 1596 99-4956-236 A G A 5530 9461 1597 99-4966-298 A G A 5531 9462 1598 99-4968-273 C T S 5532 9463 1599 99-5016-206 C T S 5533 9464 1600 99-5029-240 G T A 5534 9465 1601 99-5032-232 A C S 5535 9466 1602 99-5036-40 C T S 5536 9467 1603 99-5038-181 C T S 5537 9468 1604 99-5043-111 A C S 5538 9469 1605 99-5099-245 A G A 5539 9470 1606 99-5101-284 G T A 5540 9471 1607 99-5104-160 A G A 5541 9472 1608 99-5107-184 G T A 5542 9473 1609 99-5108-144 A G A 5543 9474 1610 99-511-33 C T S 5544 9475 1611 99-5130-355 C T S 5545 9476 1612 99-5142-74 C T S 5546 9477 1613 99-5148-269 G C S 5547 9478 1614 99-5149-436 C T S 5548 9479 1615 99-5157-422 G C S 5549 9480 1616 99-5162461 C T S 5550 9481 1617 99-5168-220 C T S 5551 9482 1618 99-5184-146 A G A 5552 9483 1619 99-5186-455 A G A 5553 9484 1620 99-5189-412 C T S 5554 9485 1621 99-5193-430 A G A 5555 9486 1622 99-5194-145 A G A 5556 9487 1623 99-5199-108 A T S 5557 9488 1624 99-5202-145 A G A 5558 9489 1625 99-5224-293 G C S 5559 9490 1626 99-5225-198 C T S 5560 9491 1627 99-5226-215 A G A 5561 9492 1628 99-5247-158 A G A 5562 9493 1629 99-5252-252 A G A 5563 9494 1630 99-5265-288 C T S 5564 9495 1631 99-5290-322 C T S 5565 9496 1632 99-5291-331 A C S 5566 9497 1633 99-5294-362 A C S 5567 9498 1634 99-5306-93 C T S 5568 9499 1635 99-5308-341 G C S 5569 9500 1636 99-5312-273 C T S 5570 9501 1637 99-5326-332 A G A 5571 9502 1638 99-5338-151 C T S 5572 9503 1639 99-5355-165 A G A 5573 9504 1640 99-5356-100 A T S 5574 9505 1641 99-5360-151 C T S 5575 9506 1642 99-5362-203 C T S 5576 9507 1643 99-5364-95 C T A 5577 9508 1644 99-5379-158 A G A 5578 9509 1645 99-5386-85 A G A 5579 9510 1646 99-5389-409 A G A 5580 9511 1647 99-5390-375 C T S 5581 9512 1648 99-5401-280 C T S 5582 9513 1649 99-5405-376 C T S 5583 9514 1650 99-5406-299 A T S 5584 9515 1651 99-5407-173 C T S 5585 9516 1652 99-5411-378 C T S 5586 9517 1653 99-5416-137 A G A 5587 9518 1654 99-5420-425 G C S 5588 9519 1655 99-5427-466 A G A 5589 9520 1656 99-5432-391 G C S 5590 9521 1657 99-5433-45 C T S 5591 9522 1658 99-5437-159 A G A 5592 9523 1659 99-5438-70 C T S 5593 9524 1660 99-5441-287 C T S 5594 9525 1661 99-5446-303 C T S 5595 9526 1662 99-5447-322 A G A 5596 9527 1663 99-5458-203 C T S 5597 9528 1664 99-5468-319 A C S 5598 9529 1665 99-5472-290 C T S 5599 9530 1666 99-5475-455 A G A 5600 9531 1667 99-5477-207 A C S 5601 9532 1668 99-5485-325 A G A 5602 9533 1669 99-5490-368 C T S 5603 9534 1670 99-5494-205 A G A 5604 9535 1671 99-5502-433 A C S 5605 9536 1672 99-5505-226 C T S 5606 9537 1673 99-5516-121 C T S 5607 9538 1674 99-5526-334 C T S 5608 9539 1675 99-5566-131 A G A 5609 9540 1676 99-5582-71 G C S 5610 9541 1677 99-5590-99 C T S 5611 9542 1678 99-5595-380 A G A 5612 9543 1679 99-5596-216 A G A 5613 9544 1680 99-5604-376 A G A 5614 9545 1681 99-5608-324 A G A 5615 9546 1682 99-5632-425 A G A 5616 9547 1683 99-5633-334 A G A 5617 9548 1684 99-5634-426 C T S 5618 9549 1685 99-5636-198 C T S 5619 9550 1686 99-5660-265 G C S 5620 9551 1687 99-5670-264 C T S 5621 9552 1688 99-5678-321 A G A 5622 9553 1689 99-5680-109 A G A 5623 9554 1690 99-5681-81 C T S 5624 9555 1691 99-5685-274 A T S 5625 9556 1692 99-5686-274 G C S 5626 9557 1693 99-5700-142 G C S 5627 9558 1694 99-5702-192 C T S 5628 9559 1695 99-5703-72 C T S 5629 9560 1696 99-5709-80 A G A 5630 9561 1697 99-5711-206 C T S 5631 9562 1698 99-5712-123 A T S 5632 9563 1699 99-5727-77 C T S 5633 9564 1700 99-5729-370 A G A 5634 9565 1701 99-5731-450 G T A 5635 9566 1702 99-5741-59 A G A 5636 9567 1703 99-5742-337 C T S 5637 9568 1704 99-5745-256 G C S 5638 9569 1705 99-5756-233 A G A 5639 9570 1706 99-576-421 G C S 5640 9571 1707 99-5760-164 A G A 5641 9572 1708 99-5770-275 C T S 5642 9573 1709 99-5781-110 G C S 5643 9574 1710 99-5795-234 A C S 5644 9575 1711 99-5813-34 A T S 5645 9576 1712 99-582-132 A G S 5646 9577 1713 99-5832-136 C T S 5647 9578 1714 99-5836-327 C T S 5648 9579 1715 99-5837-407 C T S 5649 9580 1716 99-5860-278 A G A 5650 9581 1717 99-5867-284 G T A 5651 9582 1718 99-5875-411 A G A 5652 9583 1719 99-5893-211 A G A 5653 9584 1720 99-5907-143 C T S 5654 9585 1721 99-5908-225 A G A 5655 9586 1722 99-5909-292 C T S 5656 9587 1723 99-5912-49 A G A 5657 9588 1724 99-5915-378 A G A 5658 9589 1725 99-5951-438 C T S 5659 9590 1726 99-5957-123 G C S 5660 9591 1727 99-596-228 G C S 5661 9592 1728 99-5968-382 A T S 5662 9593 1729 99-5979-96 C T S 5663 9594 1730 99-598-130 A G A 5664 9595 1731 99-6007-246 A C S 5665 9596 1732 99-6012-220 G T A 5666 9597 1733 99-602-258 A G A 5667 9598 1734 99-6038-286 A G S 5668 9599 1735 99-6042-134 G C S 5669 9600 1736 99-6051-251 G C S 5670 9601 1737 99-6067-247 C T S 5671 9602 1738 99-6069-41 A G A 5672 9603 1739 99-607-397 A G A 5673 9604 1740 99-6077-346 C T S 5674 9605 1741 99-6079-343 A G A 5675 9606 1742 99-608-183 G T A 5676 9607 1743 99-6080-99 C T S 5677 9608 1744 99-609-225 A T S 5678 9609 1745 99-6091-305 A G A 5679 9610 1746 99-6094-223 C T S 5680 9611 1747 99-6095-316 A G A 5681 9612 1748 99-6097-202 G T A 5682 9613 1749 99-610-250 A G A 5683 9614 1750 99-6112-275 C T S 5684 9615 1751 99-6117-221 A G A 5685 9616 1752 99-6122-100 A G A 5686 9617 1753 99-6131-166 A G A 5687 9618 1754 99-6135-319 C T S 5688 9619 1755 99-614-346 G C S 5689 9620 1756 99-6141-339 C T S 5690 9621 1757 99-615-387 A C S 5691 9622 1758 99-616-338 A G A 5692 9623 1759 99-6176-96 A G A 5693 9624 1760 99-6180-389 G T A 5694 9625 1761 99-6181-328 A C S 5695 9626 1762 99-6189-224 A G A 5696 9627 1763 99-619-141 C T S 5697 9628 1764 99-6191-252 A C S 5698 9629 1765 99-6193-88 C T S 5699 9630 1766 99-621-215 A G A 5700 9631 1767 99-6217-420 C T S 5701 9632 1768 99-622-95 A G A 5702 9633 1769 99-6253-308 C T S 5703 9634 1770 99-6257-226 C T S 5704 9635 1771 99-6261-172 A C S 5705 9636 1772 99-6278-391 C T S 5706 9637 1773 99-6294-184 C T S 5707 9638 1774 99-6298-280 A C S 5708 9639 1775 99-6300-106 G C S 5709 9640 1776 99-6310-217 G C S 5710 9641 1777 99-632-173 C T S 5711 9642 1778 99-6327-270 A C S 5712 9643 1779 99-6332-143 C T S 5713 9644 1780 99-6367-268 A C S 5714 9645 1781 99-6368-426 A G A 5715 9646 1782 99-6404-147 A G A 5716 9647 1783 99-6409-62 C T S 5717 9648 1784 99-6411-93 A G A 5718 9649 1785 99-6413-369 C T S 5719 9650 1786 99-6415-279 C T S 5720 9651 1787 99-6421-210 C T S 5721 9652 1788 99-6423-90 G C S 5722 9653 1789 99-6426-413 A G A 5723 9654 1790 99-6427-190 A G A 5724 9655 1791 99-6435-343 A G A 5725 9656 1792 99-6437-77 A C S 5726 9657 1793 99-6440-318 G C S 5727 9658 1794 99-6447-178 C T S 5728 9659 1795 99-6456-165 C T S 5729 9660 1796 99-6459-201 A G A 5730 9661 1797 99-646-271 C T S 5731 9662 1798 99-6463-348 A C S 5732 9663 1799 99-6468-288 A G A 5733 9664 1800 99-6478-358 A G A 5734 9665 1801 99-6480-440 A G A 5735 9666 1802 99-6489-237 G T A 5736 9667 1803 99-649-422 A T S 5737 9668 1804 99-6496-340 A G A 5738 9669 1805 99-6511-176 C T S 5739 9670 1806 99-6525-196 C T S 5740 9671 1807 99-6527-95 A C S 5741 9672 1808 99-6529-519 C T S 5742 9673 1809 99-6539-298 A G A 5743 9674 1810 99-6557-401 A G A 5744 9675 1811 99-658-367 A G A 5745 9676 1812 99-6581-45 C T S 5746 9677 1813 99-6586-359 A G A 5747 9678 1814 99-6588-94 C T S 5748 9679 1815 99-6595-322 G C S 5749 9680 1816 99-6609-103 C T S 5750 9681 1817 99-6612-185 G T A 5751 9682 1818 99-6613-223 G C S 5752 9683 1819 99-6620-294 A C S 5753 9684 1820 99-6628-474 A C S 5754 9685 1821 99-6639-290 C T S 5755 9686 1822 99-6640-342 A T S 5756 9687 1823 99-6646-465 A G A 5757 9688 1824 99-6667-63 C T S 5758 9689 1825 99-6672-314 G T A 5759 9690 1826 99-6675-324 A G A 5760 9691 1827 99-6688-363 A G A 5761 9692 1828 99-669-291 A C S 5762 9693 1829 99-6697-80 A T S 5763 9694 1830 99-670-274 A G A 5764 9695 1831 99-6705-101 C T S 5765 9696 1832 99-6706-308 C T S 5766 9697 1833 99-6715-439 C T S 5767 9698 1834 99-6726-341 A T S 5768 9699 1835 99-6730-356 A G A 5769 9700 1836 99-6753-79 A C S 5770 9701 1837 99-6757-288 G T A 5771 9702 1838 99-676-357 A G A 5772 9703 1839 99-6781-263 G T A 5773 9704 1840 99-6790-378 C T S 5774 9705 1841 99-680-228 A G A 5775 9706 1842 99-6804-426 A G A 5776 9707 1843 99-6815-484 G C S 5777 9708 1844 99-6820-251 A G A 5778 9709 1845 99-6832-178 C T S 5779 9710 1846 99-6856-433 A G A 5780 9711 1847 99-6865-455 C T S 5781 9712 1848 99-6866-130 A G A 5782 9713 1849 99-6869-256 A G A 5783 9714 1850 99-6876-229 C T S 5784 9715 1851 99-689-219 C T S 5785 9716 1852 99-6893-392 C T S 5786 9717 1853 99-6895-144 A T S 5787 9718 1854 99-6938-347 G T S 5788 9719 1855 99-694-236 G T A 5789 9720 1856 99-6940-464 A G A 5790 9721 1857 99-6942-313 A C S 5791 9722 1858 99-6951-410 G T A 5792 9723 1859 99-6956-58 C T S 5793 9724 1860 99-6957-137 G C S S794 9725 1861 99-6960-412 A T S 5795 9726 1862 99-6962-34 G C S 5796 9727 1863 99-6979-64 C T S 5797 9728 1864 99-6986-157 A G A 5798 9729 1865 99-6988-236 A C S 5799 9730 1866 99-6989-397 A G A 5800 9731 1867 99-6996-217 C T S 5801 9732 1868 99-700-123 A G A 5802 9733 1869 99-7000-235 G C S 5803 9734 1870 99-7004-304 A G A 5804 9735 1871 99-7013-250 C T S 5805 9736 1872 99-7024-122 G T A 5806 9737 1873 99-7025-226 C T S 5807 9738 1874 99-7026-247 C T S 5808 9739 1875 99-7047-225 C T S 5809 9740 1876 99-7056-49 A G A 5810 9741 1877 99-708-243 C T S 5811 9742 1878 99-7084-187 C T S 5812 9743 1879 99-7090-294 A G A 5813 9744 1880 99-7093-36 A C S 5814 9745 1881 99-7098-382 A G A 5815 9746 1882 99-7103-155 C T S 5816 9747 1883 99-7104-187 A G A 5817 9748 1884 99-7107-143 A G A 5818 9749 1885 99-7114-31 A G A 5819 9750 1886 99-7119-278 A G A 5820 9751 1887 99-7129-335 A C S 5821 9752 1888 99-7131-259 C T S 5822 9753 1889 99-7136-329 C T S 5823 9754 1890 99-7137-420 C T S 5824 9755 1891 99-7140-355 A G A 5825 9756 1892 99-7141-395 A G A 5826 9757 1893 99-7144-261 C j S 5827 9758 1894 99-7148-262 C T S 5828 9759 1895 99-7167-438 A G A 5829 9760 1896 99-7172-441 A G A 5830 9761 1897 99-7177-81 C T S 5831 9762 1898 99-718-261 A G A 5832 9763 1899 99-7183-338 A G A 5833 9764 1900 99-7193-228 G C S 5834 9765 1901 99-72-109 C T S 5835 9766 1902 99-7212-346 C T S 5836 9767 1903 99-7214-109 C T S 5837 9768 1904 99-7218-444 C T S 5838 9769 1905 99-7234-101 A T S 5839 9770 1906 99-724-246 A G A 5840 9771 1907 99-7252-279 C T S 5841 9772 1908 99-7274-172 C T S 5842 9773 1909 99-7275-150 C T S 5843 9774 1910 99-7276-286 A G A 5844 9775 1911 99-7293-201 A G A 5845 9776 1912 99-73-140 C T S 5846 9777 1913 99-7311-179 A G A 5847 9778 1914 99-7312-177 A G A 5848 9779 1915 99-7323-178 A C S 5849 9780 1916 99-7326-94 G C S 5850 9781 1917 99-7334-350 C T S 5851 9782 1918 99-734-126 C T S 5852 9783 1919 99-7349-384 G C S 5853 9784 1920 99-7356-176 A G A 5854 9785 1921 99-7363-474 C T S 5855 9786 1922 99-737-372 A G A 5856 9787 1923 99-7373-339 A G A 5857 9788 1924 99-7374-230 A G A 5858 9789 1925 99-7375-210 C T S 5859 9790 1926 99-7376-157 A T S 5860 9791 1927 99-7380-255 A G A 5861 9792 1928 99-7387-414 C T S 5862 9793 1929 99-7391-356 C T S 5863 9794 1930 99-7396-228 A T S 5864 9795 1931 99-7402-110 C T S 5865 9796 1932 99-7405-92 C T S 5866 9797 1933 99-7406-380 A G A 5867 9798 1934 99-7417-440 C T S 5868 9799 1935 99-7429-204 G T A 5869 9800 1936 99-7447-281 G C S 5870 9801 1937 99-7453-405 C T S 5871 9802 1938 99-7454-35 G C S 5872 9803 1939 99-747-252 A G A 5873 9804 1940 99-7475-179 G T A 5874 9805 1941 99-7480-66 A G A 5875 9806 1942 99-7492-275 C T S 5876 9807 1943 99-7493-249 G C S 5877 9808 1944 99-7502-382 A T S 5878 9809 1945 99-7504-342 A C S 5879 9810 1946 99-7520-222 A G A 5880 9811 1947 99-7524-130 A G A 5881 9812 1948 99-7543-467 A G A 5882 9813 1949 99-755-83 A T S 5883 9814 1950 99-7598-388 A G A 5884 9815 1951 99-760-261 C T S 5885 9816 1952 99-7604-309 A G A 5886 9817 1953 99-7605-62 G C S 5887 9818 1954 99-7608-388 C T S 5888 9819 1955 99-7610-444 C T S 5889 9820 1956 99-7611-156 A G A 5890 9821 1957 99-7614-28 A T S 5891 9822 1958 99-763-240 A G A 5892 9823 1959 99-7642-191 A G A 5893 9824 1960 99-7643-350 C T S 5894 9825 1961 99-7650-187 G C S 5895 9826 1962 99-7671-33 G C S 5896 9827 1963 99-7677-107 C T S 5897 9828 1964 99-7688-325 C T S 5898 9829 1965 99-7692-340 A G A 5899 9830 1966 99-77-318 A C A 5900 9831 1967 99-7706-303 A G A 5901 9832 1968 99-771-391 A G A 5902 9833 1969 99-7710-318 C T S 5903 9834 1970 99-7712-176 C T S 5904 9835 1971 99-7721-379 A C S 5905 9836 1972 99-7727-65 C T S 5906 9837 1973 99-7728-334 A G A 5907 9838 1974 99-7732-122 C T S 5908 9839 1975 99-7737-264 A G A 5909 9840 1976 99-7744-255 G C S 5910 9841 1977 99-7745-305 G C S 5911 9842 1978 99-7749-123 C T S 5912 9843 1979 99-7751-450 A G A 5913 9844 1980 99-7753-199 G C S 5914 9845 1981 99-7754-119 G T A 5915 9846 1982 99-7759-63 G T A 5916 9847 1983 99-7762-227 A G A 5917 9848 1984 99-7764-161 A G A 5918 9849 1985 99-7775-313 C T A 5919 9850 1986 99-7784-31 C T S 5920 9851 1987 99-7789-404 G T A 5921 9852 1988 99-7792-173 C T S 5922 9853 1989 99-7796-130 G C S 5923 9854 1990 99-7803-253 A G A 5924 9855 1991 99-781-64 A G A 5925 9856 1992 99-7840-281 A C S 5926 9857 1993 99-785-360 A G A 5927 9858 1994 99-7868-204 A G A 5928 9859 1995 99-7869-135 G C S 5929 9860 1996 99-7870-316 A C S 5930 9861 1997 99-7877-363 A G A 5931 9862 1998 99-7882-43 A G A 5932 9863 1999 99-7883-411 G C S 5933 9864 2000 99-7884-151 G T A 5934 9865 2001 99-7893-226 A G A 5935 9866 2002 99-7898-43 A T S 5936 9867 2003 99-7900-452 A G A 5937 9868 2004 99-791-236 C T S 5938 9869 2005 99-7917-429 A C S 5939 9870 2006 99-794-393 G C S 5940 9871 2007 99-7949-301 A G A 5941 9872 2008 99-795-211 C T S 5942 9873 2009 99-7967-152 C T S 5943 9874 2010 99-797-238 C T S 5944 9875 2011 99-7985-178 G C S 5945 9876 2012 99-7988-389 C T S 5946 9877 2013 99-8002-49 A G A 5947 9878 2014 99-8010-124 C T S 5948 9879 2015 99-8012-420 A G A 5949 9880 2016 99-8013-122 A G A 5950 9881 2017 99-8016-267 A G A 5951 9882 2018 99-8025-306 A C S 5952 9883 2019 99-8027-265 C T S 5953 9884 2020 99-8028-87 A T S 5954 9885 2021 99-8030-411 G C S 5955 9886 2022 99-8046-263 A G A 5956 9887 2023 99-8051-125 A G A 5957 9888 2024 99-806-152 C T S 5958 9889 2025 99-8063-174 C T S 5959 9890 2026 99-8067-79 A G A 5960 9891 2027 99-8068-258 C T S 5961 9892 2028 99-8081-340 C T S 5962 9893 2029 99-8088-247 A C S 5963 9894 2030 99-8089-246 A G A 5964 9895 2031 99-8095-164 G T A 5965 9896 2032 99-81-227 C T S 5966 9897 2033 99-810-117 A G A 5967 9898 2034 99-8102-124 C T S 5968 9899 2035 99-8120-354 A G A 5969 9900 2036 99-8128-302 C T S 5970 9901 2037 99-8141-65 A C S 5971 9902 2038 99-8161-230 A G A 5972 9903 2039 99-8162-210 C T S 5973 9904 2040 99-8164-397 A G S 5974 9905 2041 99-8170-163 C T S 5975 9906 2042 99-8173-352 C T S 5976 9907 2043 99-8181-228 A T S 5977 9908 2044 99-8186-76 G T A 5978 9909 2045 99-8188-369 G T A 5979 9910 2046 99-8192-168 C T S 5980 9911 2047 99-8219-373 A G A 5981 9912 2048 99-8245-192 G C S 5982 9913 2049 99-8255-365 A G A 5983 9914 2050 99-8256-148 A G A 5984 9915 2051 99-8266-393 A G A 5985 9916 2052 99-827-359 C T S 5986 9917 2053 99-8272-122 G C S 5987 9918 2054 99-8276-65 A C S 5988 9919 2055 99-8278-412 A G A 5989 9920 2056 99-8279-252 A T S 5990 9921 2057 99-828-259 C T S 5991 9922 2058 99-8287-122 A C S 5992 9923 2059 99-8289-179 G C S 5993 9924 2060 99-8290-174 A G A 5994 9925 2061 99-8292-240 A G A 5995 9926 2062 99-8294-408 A G A 5996 9927 2063 99-8308-237 A C S 5997 9928 2064 99-8313-107 A G A 5998 9929 2065 99-8318-50 A G A 5999 9930 2066 99-8328-298 C T S 6000 9931 2067 99-8338-212 G C S 6001 9932 2068 99-8340-364 C T S 6002 9933 2069 99-8341-99 A G A 6003 9934 2070 99-8342-33 A C S 6004 9935 2071 99-8353-291 A C S 6005 9936 2072 99-8360-401 C T S 6006 9937 2073 99-8361-103 C T S 6007 9938 2074 99-8367-239 C T S 6008 9939 2075 99-8369-276 G T A 6009 9940 2076 99-8377-429 C T S 6010 9941 2077 99-8378-69 A G A 6011 9942 2078 99-8379-337 C T S 6012 9943 2079 99-8381-114 C T S 6013 9944 2080 99-8383-158 G T A 6014 9945 2081 99-8385-244 A G A 6015 9946 2082 99-840-68 A C S 6016 9947 2083 99-8402-113 G C S 6017 9948 2084 99-8414-183 A T S 6018 9949 2085 99-8441-298 A C S 6019 9950 2086 99-8442-95 C T S 6020 9951 2087 99-8453-358 A G A 6021 9952 2088 99-8454-152 C T S 6022 9953 2089 99-8456-266 A C S 6023 9954 2090 99-8457-239 C T S 6024 9955 2091 99-8470-275 A G A 6025 9956 2092 99-8472-152 G C S 6026 9957 2093 99-8476-216 A G A 6027 9958 2094 99-8478-385 G T A 6028 9959 2095 99-8487-245 C T S 6029 9960 2096 99-8491-339 A G A 6030 9961 2097 99-8499-107 C T S 6031 9962 2098 99-8505-269 A G A 6032 9963 2099 99-851-237 C T S 6033 9964 2100 99-8510-44 A C S 6034 9965 2101 99-8514-434 C T S 6035 9966 2102 99-8530-209 A G A 6036 9967 2103 99-854-415 C T S 6037 9968 2104 99-8546-116 C T S 6038 9969 2105 99-8571-396 A G A 6039 9970 2106 99-8575-401 C T S 6040 9971 2107 99-8576-321 A G A 6041 9972 2108 99-8578-407 A C S 6042 9973 2109 99-8581-443 C T S 6043 9974 2110 99-8583-146 A C S 6044 9975 2111 99-8588-369 A G A 6045 9976 2112 99-8590-287 A G A 6046 9977 2113 99-860-419 C T S 6047 9978 2114 99-8600-393 A T S 6048 9979 2115 99-8609-434 G T A 6049 9980 2116 99-8611-383 A G A 6050 9981 2117 99-862-233 A C S 6051 9982 2118 99-8626-133 C T S 6052 9983 2119 99-8632-413 A G A 6053 9984 2120 99-8638-107 C T S 6054 9985 2121 99-8641-418 C T S 6055 9986 2122 99-8648-169 A T S 6056 9987 2123 99-8654-157 A G A 6057 9988 2124 99-8655-77 C T S 6058 9989 2125 99-8658-168 A G A 6059 9990 2126 99-866-160 C T S 6060 9991 2127 99-8662-192 A C S 6061 9992 2128 99-8663-39 C T S 6062 9993 2129 99-8665-182 A C S 6063 9994 2130 99-8671-143 A G A 6064 9995 2131 99-8695-147 A T S 6065 9996 2132 99-870-379 C T S 6066 9997 2133 99-8703-42 C T S 6067 9998 2134 99-8715-315 A G A 6068 9999 2135 99-8725-240 A G A 6069 10000 2136 99-8732-105 A G A 6070 10001 2137 99-8744-283 A G A 6071 10002 2138 99-8748-239 C T S 6072 10003 2139 99-8755-402 C T S 6073 10004 2140 99-8761-163 A G A 6074 10005 2141 99-8775-410 C T S 6075 10006 2142 99-8778-416 A G A 6076 10007 2143 99-8780-454 C T S 6077 10008 2144 99-8796-142 G T A 6078 10009 2145 99-8799-211 A G A 6079 10010 2146 99-88-216 A G S 6080 10011 2147 99-8800-250 C T S 6081 10012 2148 99-8802-119 A G A 6082 10013 2149 99-8804-83 A C S 6083 10014 2150 99-8812-220 A G A 6084 10015 2151 99-8827-400 C T S 6085 10016 2152 99-8831-41 C T S 6086 10017 2153 99-8835-400 A G A 6087 10018 2154 99-8849-167 A G A 6088 10019 2155 99-8857-96 A T S 6089 10020 2156 99-8866-150 A G S 6090 10021 2157 99-8867-278 A T S 6091 10022 2158 99-8872-391 A T S 6092 10023 2159 99-8885-447 A G A 6093 10024 2160 99-8887-397 A G A 6094 10025 2161 99-8894-123 A G A 6095 10026 2162 99-8895-272 A T S 6096 10027 2163 99-8901-283 A G A 6097 10028 2164 99-8905-184 G C S 6098 10029 2165 99-8910-170 C T S 6099 10030 2166 99-8923-138 C T S 6100 10031 2167 99-8924-415 A T S 6101 10032 2168 99-8960-426 A C S 6102 10033 2169 99-8963-409 A C S 6103 10034 2170 99-8974-386 C T S 6104 10035 2171 99-8978-52 A G A 6105 10036 2172 99-8992-43 A G A 6106 10037 2173 99-9015-255 A G A 6107 10038 2174 99-9020-110 G T A 6108 10039 2175 99-9026-273 A G A 6109 10040 2176 99-9029-132 A G A 6110 10041 2177 99-9047-183 A G A 6111 10042 2178 99-9053-311 A C S 6112 10043 2179 99-9059-197 A T S 6113 10044 2180 99-9061-309 A G A 6114 10045 2181 99-9064-194 A G A 6115 10046 2182 99-9079-158 A G A 6116 10047 2183 99-9084-200 A G A 6117 10048 2184 99-9092-167 A G A 6118 10049 2185 99-9097-342 A G A 6119 10050 2186 99-9105-68 G T A 6120 10051 2187 99-9118-393 C T S 6121 10052 2188 99-9120-197 C T S 6122 10053 2189 99-9126-25 G C S 6123 10054 2190 99-913-140 A G A 6124 10055 2191 99-9141-307 G C S 6125 10056 2192 99-9152-154 C T S 6126 10057 2193 99-9157-329 A C S 6127 10058 2194 99-9175-329 A G A 6128 10059 2195 99-9204-245 C T S 6129 10060 2196 99-921-285 C T S 6130 10061 2197 99-924-93 A G A 6131 10062 2198 99-9240-109 C T S 6132 10063 2199 99-9250-450 G T A 6133 10064 2200 99-9254-404 A G A 6134 10065 2201 99-926-98 G T A 6135 10066 2202 99-9263-283 A G A 6136 10067 2203 99-9271-70 G T A 6137 10068 2204 99-9274-246 C T S 6138 10069 2205 99-9276-163 C T S 6139 10070 2206 99-9355-134 C T S 6140 10071 2207 99-9368-223 A G A 6141 10072 2208 99-937-125 A T S 6142 10073 2209 99-9372-298 A C S 6143 10074 2210 99-9381-429 A G A 6144 10075 2211 99-9385-387 C T S 6145 10076 2212 99-9389-363 A G A 6146 10077 2213 99-9395-133 C T S 6147 10078 2214 99-9401-80 A G A 6148 10079 2215 99-9402-263 C T S 6149 10080 2216 99-9404-338 A T S 6150 10081 2217 99-9405-421 A G A 6151 10082 2218 99-941-265 A T S 6152 10083 2219 99-9410-205 C T S 6153 10084 2220 99-9412-202 C T S 6154 10085 2221 99-9417-151 C T S 6155 10086 2222 99-942-381 C T S 6156 10087 2223 99-9420-318 C T S 6157 10088 2224 99-9421-51 A G A 6158 10089 2225 99-9422-41 A G A 6159 10090 2226 99-9423-394 A G A 6160 10091 2227 99-9424-229 A G A 6161 10092 2228 99-9427-454 C T S 6162 10093 2229 99-9446-394 A G A 6163 10094 2230 99-9448-292 C T S 6164 10095 2231 99-9462-362 A G A 6165 10096 2232 99-9471-230 A G A 6166 10097 2233 99-949-214 A G A 6167 10098 2234 99-9491-388 A G A 6168 10099 2235 99-9493-455 G C S 6169 10100 2236 99-9499-111 A T S 6170 10101 2237 99-950-418 C T S 6171 10102 2238 99-9513-285 A G A 6172 10103 2239 99-952-252 G C S 6173 10104 2240 99-9527-211 C T S 6174 10105 2241 99-9531-340 C T S 6175 10106 2242 99-9538-395 A G A 6176 10107 2243 99-954-45 A C S 6177 10108 2244 99-9542-164 G T A 6178 10109 2245 99-9545-100 A C S 6179 10110 2246 99-9554-345 A C S 6180 10111 2247 99-9555-348 C T S 6181 10112 2248 99-9556-349 A G A 6182 10113 2249 99-9567-229 C T A 6183 10114 2250 99-9572-240 C T A 6184 10115 2251 99-9577-284 C T A 6185 10116 2252 99-9579-363 G C S 6186 10117 2253 99-958-92 C T A 6187 10118 2254 99-9587-338 A G S 6188 10119 2255 99-961-150 C T S 6189 10120 2256 99-963-395 A G A 6190 10121 2257 99-965-165 C T S 6191 10122 2258 99-967-306 C T S 6192 10123 2259 99-976-246 C T S 6193 10124 2260 99-979-343 A C S 6194 10125 2261 99-10000-518 G A S 6195 10126 2262 99-10016-115 T A S 6196 10127 2263 99-10027-378 G A S 6197 10128 2264 99-10028-93 C G S 6198 10129 2265 99-10031-130 T C A 6199 10130 2266 99-10046-199 T C S 6200 10131 2267 99-10064-252 T C S 6201 10132 2268 99-10066-465 G T A 6202 10133 2269 99-10067-168 G A A 6203 10134 2270 99-10078-341 T C S 6204 10135 2271 99-10104-464 C A S 6205 10136 2272 99-10106-247 G T A 6206 10137 2273 99-10108-419 G A S 6207 10138 2274 99-10118-323 T C A 6208 10139 2275 99-10126-413 C T A 6209 10140 2276 99-10127-506 A T S 6210 10141 2277 99-10137-195 T G A 6211 10142 2278 99-10142-293 G A S 6212 10143 2279 99-10143-111 A G S 6213 10144 2280 99-10146-202 T A S 6214 10145 2281 99-10149-291 T C A 6215 10146 2282 99-10151-340 G A S 6216 10147 2283 99-10153-267 A G S 6217 10148 2284 99-10155423 C G S 6218 10149 2285 99-10173-122 A G S 6219 10150 2286 99-10179-48 G A S 6220 10151 2287 99-1018-244 A C A 6221 10152 2288 99-10183-166 G T A 6222 10153 2289 99-10185-402 C A S 6223 10154 2290 99-10188-116 G A S 6224 10155 2291 99-10201-115 C T A 6225 10156 2292 99-10207-173 A G S 6226 10157 2293 99-10211-380 A G S 6227 10158 2294 99-10216-336 A C S 6228 10159 2295 99-10220-312 C A S 6229 10160 2296 99-10223-153 T A S 6230 10161 2297 99-10224-223 T A S 6231 10162 2298 99-10234-334 A G S 6232 10163 2299 99-1024-403 G A S 6233 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T A 6262 10193 2329 99-10473-259 T C A 6263 10194 2330 99-10474-223 G C S 6264 10195 2331 99-10481-217 C T A 6265 10196 2332 99-10487-57 T C A 6266 10197 2333 99-10488-146 G C S 6267 10198 2334 99-10491-300 C T A 6268 10199 2335 99-10499-102 T G A 6269 10200 2336 99-10502-161 T C A 6270 10201 2337 99-10506-307 A C S 6271 10202 2338 99-10507-216 C T A 6272 10203 2339 99-10509-122 T G A 6273 10204 2340 99-1051-284 G A A 6274 10205 2341 99-10513-347 T C A 6275 10206 2342 99-10514-546 G T A 6276 10207 2343 99-10521-296 C T A 6277 10208 2344 99-10522-395 A C A 6278 10209 2345 99-10536-90 C A S 6279 10210 2346 99-10539-208 G A S 6280 10211 2347 99-10542-326 C T A 6281 10212 2348 99-10543-278 A T S 6282 10213 2349 99-1055-140 C T S 6283 10214 2350 99-10557-276 T C A 6284 10215 2351 99-10567-233 A G S 6285 10216 2352 99-10570-107 G A S 6286 10217 2353 99-10573-375 G A S 6287 10218 2354 99-10575-416 T C A 6288 10219 2355 99-10576-351 A G S 6289 10220 2356 99-10577-36 T C A 6290 10221 2357 99-10581-354 G C S 6291 10222 2358 99-10589-360 A C S 6292 10223 2359 99-10601-463 C G S 6293 10224 2360 99-10606-92 G A S 6294 10225 2361 99-10608-353 C T A 6295 10226 2362 99-10613-277 C G S 6296 10227 2363 99-10618-404 A T S 6297 10228 2364 99-10626-196 G C S 6298 10229 2365 99-10630-236 G A S 6299 10230 2366 99-10632-55 A T S 6300 10231 2367 99-10634-141 G A S 6301 10232 2368 99-10643-161 T C A 6302 10233 2369 99-10659-208 C T A 6303 10234 2370 99-10661-153 G A S 6304 10235 2371 99-10662-397 C T A 6305 10236 2372 99-10667-251 G C S 6306 10237 2373 99-10675-109 A G S 6307 10238 2374 99-1068-309 C T S 6308 10239 2375 99-10683-117 A G S 6309 10240 2376 99-10689-419 A G S 6310 10241 2377 99-10692-377 G A S 6311 10242 2378 99-10694-446 C T A 6312 10243 2379 99-10695-161 C T A 6313 10244 2380 99-1070-342 C T S 6314 10245 2381 99-10702-261 G A S 6315 10246 2382 99-10706-228 T C A 6316 10247 2383 99-10708-28 G A S 6317 10248 2384 99-10709-460 G A S 6318 10249 2385 99-10715-43 A G S 6319 10250 2386 99-10719-455 C T S 6320 10251 2387 99-10720-63 A G S 6321 10252 2388 99-10731-195 C T S 6322 10253 2389 99-10735-238 T C A 6323 10254 2390 99-1074-127 G A A 6324 10255 2391 99-10741-421 A C S 6325 10256 2392 99-10743-315 T A S 6326 10257 2393 99-1075-314 G A A 6327 10258 2394 99-10752-366 A G S 6328 10259 2395 99-1076-116 C T A 6329 10260 2396 99-10769-291 A C S 6330 10261 2397 99-10771-266 A C S 6331 10262 2398 99-10775-331 A G S 6332 10263 2399 99-10776-447 T A S 6333 10264 2400 99-1079-237 C G S 6334 10265 2401 99-1081-159 A T S 6335 10266 2402 99-10816-272 G A S 6336 10267 2403 99-1082-180 T A S 6337 10268 2404 99-10839-239 C T A 6338 10269 2405 99-10842-232 A G S 6339 10270 2406 99-10843-114 A G S 6340 10271 2407 99-10856-246 C G S 6341 10272 2408 99-10861-96 T C A 6342 10273 2409 99-10862-397 T C A 6343 10274 2410 99-10864-418 C G S 6344 10275 2411 99-10870-234 G A S 6345 10276 2412 99-10874-69 G A S 6346 10277 2413 99-10879-386 A G S 6347 10278 2414 99-10887-214 A G S 6348 10279 2415 99-10890-201 T G A 6349 10280 2416 99-10894-35 T C A 6350 10281 2417 99-10898-209 G T A 6351 10282 2418 99-10904-111 C T A 6352 10283 2419 99-10905-85 C T A 6353 10284 2420 99-1091-446 C T S 6354 10285 2421 99-10927-388 G C S 6355 10286 2422 99-10929-298 T A S 6356 10287 2423 99-10930-95 A T S 6357 10288 2424 99-10937-64 T C A 6358 10289 2425 99-10944-83 G A S 6359 10290 2426 99-10951-434 T C A 6360 10291 2427 99-10959-113 C T A 6361 10292 2428 99-10964-89 T C A 6362 10293 2429 99-10965-174 C A S 6363 10294 2430 99-10966-113 G A S 6364 10295 2431 99-10974-193 T A S 6365 10296 2432 99-10978-393 T A S 6366 10297 2433 99-10979-156 T A S 6367 10298 2434 99-10988-242 G A S 6368 10299 2435 99-10992-98 G A S 6369 10300 2436 99-11000-163 A G S 6370 10301 2437 99-11001-393 C T A 6371 10302 2438 99-11003-361 G A S 6372 10303 2439 99-11006-426 C T A 6373 10304 2440 99-11007-68 T C A 6374 10305 2441 99-11014-194 C A S 6375 10306 2442 99-11034-317 C T A 6376 10307 2443 99-11035-299 C T A 6377 10308 2444 99-11037-218 C A S 6378 10309 2445 99-1105-127 A C S 6379 10310 2446 99-1J051-154 A T S 6380 10311 2447 99-11063-111 G C S 6381 10312 2448 99-11074-187 A G S 6382 10313 2449 99-11075-311 G A S 6383 10314 2450 99-11089-424 C A S 6384 10315 2451 99-11094-427 G A A 6385 10316 2452 99-11099-179 A G S 6386 10317 2453 99-11103-88 A T S 6387 10318 2454 99-11106-117 C T A 6388 10319 2455 99-11110-375 C T A 6389 10320 2456 99-11115-133 A G S 6390 10321 2457 99-11119-132 T C A 6391 10322 2458 99-11128-162 A G 8 6392 10323 2459 99-11136-374 G A S 6393 10324 2460 99-11142-139 T C A 6394 10325 2461 99-11143-443 A C S 6395 10326 2462 99-11144-137 G A S 6396 10327 2463 99-11148-369 A C S 6397 10328 2464 99-11158-255 C T A 6398 10329 2465 99-11163-293 C G S 6399 10330 2466 99-11164-298 C T A 6400 10331 2467 99-11168-197 G A S 6401 10332 2468 99-11175-348 A C S 6402 10333 2469 99-11179-239 C T A 6403 10334 2470 99-11180-148 A G S 6404 10335 2471 99-11183-166 G A S 6405 10336 2472 99-11191-86 A G S 6406 10337 2473 99-11210-235 G A S 6407 10338 2474 99-11214-188 T G A 6408 10339 2475 99-11218-174 G A S 6409 10340 2476 99-11236-63 C T A 6410 10341 2477 99-11247-86 C G S 6411 10342 2478 99-11248-404 A G S 6412 10343 2479 99-11252-263 T A S 6413 10344 2480 99-11255-375 G A S 6414 10345 2481 99-11260-422 C T A 6415 10346 2482 99-11261-255 G A S 6416 10347 2483 99-11293-125 G A S 6417 10348 2484 99-11313-95 G A S 6418 10349 2485 99-11320-29 C T A 6419 10350 2486 99-11326-356 A G S 6420 10351 2487 99-11340-89 C T A 6421 10352 2488 99-11346-222 T C A 6422 10353 2489 99-11350-116 A G S 6423 10354 2490 99-11356-187 A T S 6424 10355 2491 99-11362-334 C T A 6425 10356 2492 99-11369-112 C G S 6426 10357 2493 99-11372-162 C A 8 6427 10358 2494 99-11377-384 A G S 6428 10359 2495 99-11381-256 C G S 6429 10360 2496 99-11385-245 C T A 6430 10361 2497 99-11413-239 A G S 6431 10362 2498 99-1143-340 G A S 6432 10363 2499 99-11430-162 C A S 6433 10364 2500 99-11431-333 A G S 6434 10365 2501 99-11449-297 A G S 6435 10366 2502 99-11464-236 C G S 6436 10367 2503 99-11466-107 T C A 6437 10368 2504 99-11485-396 T A S 6438 10369 2505 99-11492-360 A T S 6439 10370 2506 99-11499-45 G A S 6440 10371 2507 99-11505-92 A G S 6441 10372 2508 99-11506-224 G C S 6442 10373 2509 99-11520-170 A G S 6443 10374 2510 99-11521-146 G T A 6444 10375 2511 99-11522-313 C T A 6445 10376 2512 99-11528-137 A T S 6446 10377 2513 99-11530-388 G A S 6447 10378 2514 99-11533-375 C G S 6448 10379 2515 99-11535-193 G T A 6449 10380 2516 99-11543-415 A T S 6450 10381 2517 99-11545-180 T C A 6451 10382 2518 99-11555-397 C T A 6452 10383 2519 99-11559-81 C A S 6453 10384 2520 99-11563-183 A G S 6454 10385 2521 99-11565-305 C A S 6455 10386 2522 99-11566-385 G A S 6456 10387 2523 99-11580-97 A G S 6457 10388 2524 99-11584-69 A G 8 6458 10389 2525 99-11587-202 A G S 6459 10390 2526 99-11592-297 C A S 6460 10391 2527 99-11600-48 A G S 6461 10392 2528 99-11601-441 C T A 6462 10393 2529 99-11602-93 G A S 6463 10394 2530 99-11604-396 T C A 6464 10395 2531 99-11611-259 A G S 6465 10396 2532 99-11613-315 T C A 6466 10397 2533 99-11620-149 A C S 6467 10398 2534 99-11635-363 C T A 6468 10399 2535 99-11643-378 C T A 6469 10400 2536 99-11645-157 C T A 6470 10401 2537 99-11658-275 G A S 6471 10402 2538 99-11668-308 T C A 6472 10403 2539 99-11669-394 C T A 6473 10404 2540 99-11670-486 G T A 6474 10405 2541 99-11685-200 T C A 6475 10406 2542 99-11697-345 C T A 6476 10407 2543 99-11700-326 T C A 6477 10408 2544 99-11704-23 T C A 6478 10409 2545 99-11705-302 G T A 6479 10410 2546 99-11723-211 A T S 6480 10411 2547 99-11743-233 C T A 6481 10412 2548 99-11745-256 A G S 6482 10413 2549 99-11746-238 A G S 6483 10414 2550 99-11780-292 G T A 6484 10415 2551 99-11785-167 A G S 6485 10416 2552 99-11786-98 G T A 6486 10417 2553 99-11787-281 G A S 6487 10418 2554 99-11788-69 G A S 6488 10419 2555 99-11789-348 A C S 6489 10420 2556 99-11797-147 A G S 6490 10421 2557 99-11810-289 A C S 6491 10422 2558 99-11811-158 A G S 6492 10423 2559 99-1182-310 G A A 6493 10424 2560 99-11823-118 G A S 6494 10425 2561 99-11824-90 T A S 6495 10426 2562 99-1183-182 G A A 6496 10427 2563 99-11830-334 C G S 6497 10428 2564 99-11831-321 C T A 6498 10429 2565 99-11839-223 C T A 6499 10430 2566 99-11842-197 T C A 6500 10431 2567 99-1185-317 T G A 6501 10432 2568 99-11851-45 C G S 6502 10433 2569 99-11857-368 G A S 6503 10434 2570 99-1186-249 G A A 6504 10435 2571 99-11861-254 C T A 6505 10436 2572 99-11877-237 T C A 6506 10437 2573 99-11880-90 G C S 6507 10438 2574 99-11882-120 A G S 6508 10439 2575 99-11894-470 G A S 6509 10440 2576 99-11917-129 A G S 6510 10441 2577 99-11922-206 G A S 6511 10442 2578 99-11930-395 C T A 6512 10443 2579 99-11966-288 A G S 6513 10444 2580 99-11989-233 C A S 6514 10445 2581 99-11993-468 C T A 6515 10446 2582 99-12000-355 T C A 6516 10447 2583 99-12005-282 C T A 6517 10448 2584 99-12017-203 G T A 6518 10449 2585 99-1202-340 C T S 6519 10450 2586 99-12028-121 T C A 6520 10451 2587 99-1203-272 A G A 6521 10452 2588 99-12038-420 C T A 6522 10453 2589 99-12039-389 C T A 6523 10454 2590 99-12048-300 A G S 6524 10455 2591 99-12049-245 A G S 6525 10456 2592 99-12050-459 A G S 6526 10457 2593 99-12061-211 A G S 6527 10458 2594 99-12062-94 G A S 6528 10459 2595 99-12068-348 T C A 6529 10460 2596 99-12087-45 C T A 6530 10461 2597 99-1211-59 C T A 6531 10462 2598 99-12130-72 G A S 6532 10463 2599 99-12133-294 T C A 6533 10464 2600 99-12135-288 C A S 6534 10465 2601 99-12152-332 A G S 6535 10466 2602 99-12158-148 A G S 6536 10467 2603 99-12168-256 C T A 6537 10468 2604 99-12171-93 G A S 6538 10469 2605 99-12178-423 C T A 6539 10470 2606 99-12181-226 T C A 6540 10471 2607 99-12186-229 C T A 6541 10472 2608 99-12198-289 A G S 6542 10473 2609 99-12199-246 T C A 6543 10474 2610 99-12203-356 C G S 6544 10475 2611 99-12224-368 A G S 6545 10476 2612 99-12228-184 C T A 6546 10477 2613 99-12241-380 T G A 6547 10478 2614 99-12253-145 T C A 6548 10479 2615 99-12265-324 A T S 6549 10480 2616 99-12267-161 G T A 6550 10481 2617 99-12268-54 G A S 6551 10482 2618 99-12270-408 T G A 6552 10483 2619 99-12271-298 G A S 6553 10484 2620 99-12275-214 A T S 6554 10485 2621 99-12299-433 T G A 6555 10486 2622 99-12303-460 C T A 6556 10487 2623 99-12335-394 C T A 6557 10488 2624 99-12338-83 T C A 6558 10489 2625 99-12344-171 G C S 6559 10490 2626 99-12347-490 G C S 6560 10491 2627 99-12348-74 T G A 6561 10492 2628 99-12352-124 T G A 6562 10493 2629 99-12356-272 T C A 6563 10494 2630 99-12361-88 T C A 6564 10495 2631 99-12368-335 A C S 6565 10496 2632 99-12370-67 G A S 6566 10497 2633 99-12384-135 G A S 6567 10498 2634 99-12388-466 G A S 6568 10499 2635 99-12393-326 A G S 6569 10500 2636 99-12399-180 C T A 6570 10501 2637 99-12412-381 T C A 6571 10502 2638 99-12415-509 A G S 6572 10503 2639 99-12444-400 A G S 6573 10504 2640 99-12465-227 G A A 6574 10505 2641 99-12468-236 G T A 6575 10506 2642 99-12470-288 G A A 6576 10507 2643 99-12522-196 T C S 6577 10508 2644 99-12561-278 C G S 6578 10509 2645 99-12570-265 G A A 6579 10510 2646 99-12595-313 C T S 6580 10511 2647 99-12596-334 T C S 6581 10512 2648 99-12598-191 C A S 6582 10513 2649 99-12602-212 G T A 6583 10514 2650 99-12605-365 C T S 6584 10515 2651 99-12607-384 A G A 6585 10516 2652 99-12664-222 C A S 6586 10517 2653 99-12696-116 T C 8 6587 10518 2654 99-12960-443 G A S 6588 10519 2655 99-12965-451 C T A 6589 10520 2656 99-12969-128 C T A 6590 10521 2657 99-12970-339 A G S 6591 10522 2658 99-12973-162 G A S 6592 10523 2659 99-13074-132 T C A 6593 10524 2660 99-13077-340 C T A 6594 10525 2661 99-1311-59 C G S 6595 10526 2662 99-13113-234 G A S 6596 10527 2663 99-13205-67 T A S 6597 10528 2664 99-1326-203 C T A 6598 10529 2665 99-1333-123 C G S 6599 10530 2666 99-1335-195 A G A 6600 10531 2667 99-13350-376 T G A 6601 10532 2668 99-13376-288 A T S 6602 10533 2669 99-13473-135 C T A 6603 10534 2670 99-13530-325 T C S 6604 10535 2671 99-13563-83 C T S 6605 10536 2672 99-13579-242 G C S 6606 10537 2673 99-13609-327 T C A 6607 10538 2674 99-13621-358 T C S 6608 10539 2675 99-1370-401 A G S 6609 10540 2676 99-13864-64 G T A 6610 10541 2677 99-13938-286 T C A 6611 10542 2678 99-13943-247 T C A 6612 10543 2679 99-13948-182 T A S 6613 10544 2680 99-13966-334 T C A 6614 10545 2681 99-14002-395 C G S 6615 10546 2682 99-14022-347 C T A 6616 10547 2683 99-14042-464 G A S 6617 10548 2684 99-14045-353 T C A 6618 10549 2685 99-14074-326 C T A 6619 10550 2686 99-14093-333 T C A 6620 10551 2687 99-14105-357 G C S 6621 10552 2688 99-14107-175 A G S 6622 10553 2689 99-14111-346 C G S 6623 10554 2690 99-14177-226 A G S 6624 10555 2691 99-14198-374 T G A 6625 10556 2692 99-14225-345 T C A 6626 10557 2693 99-14228-387 C G S 6627 10558 2694 99-14410-373 T C A 6628 10559 2695 99-14413-383 T G A 6629 10560 2696 99-14415-106 C T A 6630 10561 2697 99-14424-353 G A S 6631 10562 2698 99-14473-243 A C S 6632 10563 2699 99-14476-377 T G A 6633 10564 2700 99-14481-386 T C A 6634 10565 2701 99-14489-415 G T A 6635 10566 2702 99-14673-334 A G S 6636 10567 2703 99-14705-290 A G S 6637 10568 2704 99-14739-205 C G S 6638 10569 2705 99-14743-418 C T A 6639 10570 2706 99-14944-119 A C S 6640 10571 2707 99-14949-472 G A S 6641 10572 2708 99-15000-259 C T A 6642 10573 2709 99-15067-278 T C A 6643 10574 2710 99-15192-224 C T A 6644 10575 2711 99-15369-90 C T A 6645 10576 2712 99-15423-223 G A S 6646 10577 2713 99-15471-316 G T A 6647 10578 2714 99-15538-250 T C A 6648 10579 2715 99-15588-430 A G S 6649 10580 2716 99-15615-368 G A S 6650 10581 2717 99-15653-359 G A S 6651 10582 2718 99-15654-122 G A S 6652 10583 2719 99-15724-147 C T A 6653 10584 2720 99-15784-28 G T A 6654 10585 2721 99-1591-235 G A A 6655 10586 2722 99-15963-394 A G S 6656 10587 2723 99-15984-100 A G S 6657 10588 2724 99-16017-426 C T A 6658 10589 2725 99-16026-359 G A S 6659 10590 2726 99-1624-377 G A A 6660 10591 2727 99-16241-126 T G A 6661 10592 2728 99-16259-304 A C S 6662 10593 2729 99-16284-389 T C A 6663 10594 2730 99-16343-30 T C A 6664 10595 2731 99-16401-88 C T A 6665 10596 2732 99-16406-349 C T A 6666 10597 2733 99-16422-240 A G S 6667 10598 2734 99-16428-275 C T A 6668 10599 2735 99-16430-358 T G A 6669 10600 2736 99-16432-114 T A S 6670 10601 2737 99-16647-382 T C A 6671 10602 2738 99-16661-147 G A S 6672 10603 2739 99-16686-82 A C S 6673 10604 2740 99-16714-82 G A A 6674 10605 2741 99-16735-210 A G A 6675 10606 2742 99-16739-245 G A A 6676 10607 2743 99-16751-318 A T S 6677 10608 2744 99-16752-78 C G S 6678 10609 2745 99-16754-63 A G A 6679 10610 2746 99-16758-60 G T A 6680 10611 2747 99-16761-370 A G A 6681 10612 2748 99-16769-459 G A A 6682 10613 2749 99-16771-222 C A S 6683 10614 2750 99-16772-36 T C S 6684 10615 2751 99-16774-183 G T A 6685 10616 2752 99-16776-275 C G S 6686 10617 2753 99-16794-291 A T S 6687 10618 2754 99-16797-385 G A A 6688 10619 2755 99-16815-282 G A A 6689 10620 2756 99-16835-413 G A A 6690 10621 2757 99-16909-151 T C A 6691 10622 2758 99-17024-215 C T S 6692 10623 2759 99-17046-162 A C S 6693 10624 2760 99-17075-173 C T A 6694 10625 2761 99-17107-271 G T A 6695 10626 2762 99-17162-81 G T A 6696 10627 2763 99-17167-55 A G S 6697 10628 2764 99-17214-451 T C A 6698 10629 2765 99-17254-339 G A A 6699 10630 2766 99-17402-339 G A S 6700 10631 2767 99-17492-271 T C A 6701 10632 2768 99-17519-116 G A S 6702 10633 2769 99-17581-374 G A S 6703 10634 2770 99-17716-400 C T S 6704 10635 2771 99-18122-403 C T A 6705 10636 2772 99-18126-160 A T S 6706 10637 2773 99-18127-283 A G S 6707 10638 2774 99-18141-152 T C A 6708 10639 2775 99-18190-317 G A S 6709 10640 2776 99-18321-371 T C S 6710 10641 2777 99-1833-56 T C S 6711 10642 2778 99-18334-485 A G A 6712 10643 2779 99-18396-324 A C S 6713 10644 2780 99-18471-410 A G A 6714 10645 2781 99-18528-195 G A A 6715 10646 2782 99-18576-182 A C S 6716 10647 2783 99-18581-34 T C S 6717 10648 2784 99-18645-309 G A S 6718 10649 2785 99-18696-213 T C S 6719 10650 2786 99-18698-346 C G S 6720 10651 2787 99-18710-208 C T S 6721 10652 2788 99-18717-319 T A S 6722 10653 2789 99-18718-362 C G S 6723 10654 2790 99-18771-300 C T S 6724 10655 2791 99-1879-393 G A A 6725 10656 2792 99-18886-50 T A S 6726 10657 2793 99-18944-242 C T S 6727 10658 2794 99-19023-347 T C S 6728 10659 2795 99-19027-222 A G A 6729 10660 2796 99-19033-208 G T A 6730 10661 2797 99-19040-395 A G A 6731 10662 2798 99-19041-87 A G A 6732 10663 2799 99-19048-487 C T S 6733 10664 2800 99-19050-251 T C S 6734 10665 2801 99-19053-241 T A S 6735 10666 2802 99-19055-264 T C S 6736 10667 2803 99-19059-347 C A S 6737 10668 2804 99-19069-44 C T S 6738 10669 2805 99-19095-106 T C S 6739 10670 2806 99-19096-317 A G A 6740 10671 2807 99-19104-66 T C S 6741 10672 2808 99-19105-114 A G A 6742 10673 2809 99-19108-156 C T S 6743 10674 2810 99-19110-175 A G A 6744 10675 2811 99-19122-58 A G A 6745 10676 2812 99-19123-242 A G A 6746 10677 2813 99-19130-86 T C S 6747 10678 2814 99-19137-156 C T S 6748 10679 2815 99-19142-245 T C S 6749 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C A S 6778 10709 2845 99-19375-434 G A A 6779 10710 2846 99-19381-249 G A A 6780 10711 2847 99-19383-432 G C S 6781 10712 2848 99-19384-63 G A A 6782 10713 2849 99-19418-61 G A A 6783 10714 2850 99-19420-86 A T S 6784 10715 2851 99-19426-250 G A A 6785 10716 2852 99-19431-249 A G A 6786 10717 2853 99-19438-261 T A S 6787 10718 2854 99-19442-48 G A A 6788 10719 2855 99-19444-350 T C S 6789 10720 2856 99-19450-440 C A S 6790 10721 2857 99-19453-250 G T A 6791 10722 2858 99-19457-182 T C S 6792 10723 2859 99-19460-346 G T A 6793 10724 2860 99-19461-282 T C S 6794 10725 2861 99-19464-165 A G A 6795 10726 2862 99-19466-406 C T S 6796 10727 2863 99-19474-266 G T S 6797 10728 2864 99-19475-113 G C S 6798 10729 2865 99-19477-208 T C S 6799 10730 2866 99-19504-468 T C S 6800 10731 2867 99-19528-278 C T S 6801 10732 2868 99-19529-118 A G A 6802 10733 2869 99-19532-207 G T A 6803 10734 2870 99-19538-272 A G A 6804 10735 2871 99-19544-329 G A A 6805 10736 2872 99-19546-473 G A A 6806 10737 2873 99-19550-397 G T A 6807 10738 2874 99-19553-52 T C S 6808 10739 2875 99-19557-152 A G A 6809 10740 2876 99-19560-289 G T A 6810 10741 2877 99-19562-227 G A A 6811 10742 2878 99-19566-337 C G S 6812 10743 2879 99-19568-273 C T S 6813 10744 2880 99-19575-299 A G A 6814 10745 2881 99-19578-307 A G A 6815 10746 2882 99-19580-323 C T S 6816 10747 2883 99-19584-352 C T S 6817 10748 2884 99-19588-438 G A A 6818 10749 2885 99-19589-118 C T S 6819 10750 2886 99-19601-95 T G A 6820 10751 2887 99-19624-48 C G S 6821 10752 2888 99-19634-149 A G A 6822 10753 2889 99-19639-225 A G A 6823 10754 2890 99-19645-339 A G A 6824 10755 2891 99-19650-338 G C S 6825 10756 2892 99-19651-133 A C S 6826 10757 2893 99-19664-328 G A A 6827 10758 2894 99-19673-125 C G S 6828 10759 2895 99-19678-269 G C S 6829 10760 2896 99-19685-39 A T S 6830 10761 2897 99-19697-304 C T S 6831 10762 2898 99-19703-75 C G S 6832 10763 2899 99-19705-128 T C S 6833 10764 2900 99-19709-299 T G A 6834 10765 2901 99-19711-169 G T A 6835 10766 2902 99-19722-150 T C S 6836 10767 2903 99-19731-244 A G A 6837 10768 2904 99-19732-385 T A S 6838 10769 2905 99-19736-62 G A A 6839 10770 2906 99-19745-330 C T S 6840 10771 2907 99-19749-158 G A A 6841 10772 2908 99-19752-88 T C S 6842 10773 2909 99-19753-300 G A A 6843 10774 2910 99-19756-85 T C S 6844 10775 2911 99-19764-177 T C S 6845 10776 2912 99-19769-227 C T S 6846 10777 2913 99-19780-179 A G A 6847 10778 2914 99-19785-140 A G A 6848 10779 2915 99-19790-398 G A A 6849 10780 2916 99-19791-103 G T A 6850 10781 2917 99-19795-199 A G A 6851 10782 2918 99-19796-256 T G A 6852 10783 2919 99-19807-396 C T S 6853 10784 2920 99-19813-55 C T S 6854 10785 2921 99-19818-156 C T S 6855 10786 2922 99-19826-285 G A A 6856 10787 2923 99-19839-223 A G A 6857 10788 2924 99-19851-40 C G S 6858 10789 2925 99-19858-91 C T S 6859 10790 2926 99-19860-68 A G A 6860 10791 2927 99-19864-112 T C S 6861 10792 2928 99-19871-422 T C S 6862 10793 2929 99-19872-136 G A A 6863 10794 2930 99-19875-99 A G A 6864 10795 2931 99-19876-394 A C S 6865 10796 2932 99-19890-235 A C S 6866 10797 2933 99-19896-142 G T A 6867 10798 2934 99-19901-383 C T S 6868 10799 2935 99-19906-136 C G S 6869 10800 2936 99-19911-90 G C S 6870 10801 2937 99-19916-380 T C S 6871 10802 2938 99-19922-42 G A A 6872 10803 2939 99-19923-383 G A A 6873 10804 2940 99-19933-251 A G A 6874 10805 2941 99-19937-235 A G A 6875 10806 2942 99-19944-306 T A S 6876 10807 2943 99-19951-313 A T S 6877 10808 2944 99-20038-204 T C S 6878 10809 2945 99-20072-277 A T S 6879 10810 2946 99-20226-32 T C S 6880 10811 2947 99-20228-290 T C S 6881 10812 2948 99-20234-101 C G S 6882 10813 2949 99-20537-433 G C S 6883 10814 2950 99-20733-79 G A A 6884 10815 2951 99-20815-363 A T S 6885 10816 2952 99-20896-383 A G A 6886 10817 2953 99-20958-373 A G A 6887 10818 2954 99-21057-337 T C S 6888 10819 2955 99-21059-118 T C S 6889 10820 2956 99-21110-304 C G S 6890 10821 2957 99-21123-62 C T S 6891 10822 2958 99-21133-169 G A A 6892 10823 2959 99-21181-413 A G A 6893 10824 2960 99-21192-164 T A S 6894 10825 2961 99-21227-295 T C S 6895 10826 2962 99-21229-81 G A A 6896 10827 2963 99-21240-419 C T S 6897 10828 2964 99-21242-57 T A S 6898 10829 2965 99-21244-495 G A A 6899 10830 2966 99-21252-77 T C S 6900 10831 2967 99-21267-111 C T S 6901 10832 2968 99-21284-322 G A A 6902 10833 2969 99-21293-252 A G A 6903 10834 2970 99-21307-370 A G A 6904 10835 2971 99-21310-416 A G A 6905 10836 2972 99-21312-319 C A S 6906 10837 2973 99-21323-142 A G A 6907 10838 2974 99-21327-94 A G A 6908 10839 2975 99-21328-173 C T S 6909 10840 2976 99-21329-518 A G A 6910 10841 2977 99-21342-350 T C S 6911 10842 2978 99-21346-290 G A A 6912 10843 2979 99-21360-343 A G A 6913 10844 2980 99-21361-97 G T A 6914 10845 2981 99-21377-73 C T S 6915 10846 2982 99-21378-303 T C S 6916 10847 2983 99-21391-418 G A A 6917 10848 2984 99-21401-117 T C S 6918 10849 2985 99-21423-302 T C S 6919 10850 2986 99-21433-238 T G A 6920 10851 2987 99-21441-420 A G A 6921 10852 2988 99-21444-227 T C S 6922 10853 2989 99-21448-361 A G A 6923 10854 2990 99-21461-375 C T S 6924 10855 2991 99-21463-258 A G A 6925 10856 2992 99-21465-58 C A S 6926 10857 2993 99-21486-88 C A S 6927 10858 2994 99-21492-310 C T S 6928 10859 2995 99-21502-211 G A A 6929 10860 2996 99-21508-131 C A S 6930 10861 2997 99-21510-466 T A S 6931 10862 2998 99-21512-165 A T S 6932 10863 2999 99-21516-293 G T A 6933 10864 3000 99-21533-445 C T S 6934 10865 3001 99-21560-376 G A A 6935 10866 3002 99-21561-41 T C S 6936 10867 3003 99-21566-152 C T S 6937 10868 3004 99-21578-105 T C S 6938 10869 3005 99-21580-141 A G A 6939 10870 3006 99-21591-181 T G A 6940 10871 3007 99-21592-43 C T S 6941 10872 3008 99-21607-114 A G A 6942 10873 3009 99-21615-133 C T S 6943 10874 3010 99-21657-161 T C S 6944 10875 3011 99-21664-278 G T A 6945 10876 3012 99-21666-96 C A S 6946 10877 3013 99-21673-106 A T S 6947 10878 3014 99-21674-245 G C S 6948 10879 3015 99-21687-313 G A A 6949 10880 3016 99-21690-162 A G A 6950 10881 3017 99-21693-368 C T S 6951 10882 3018 99-21699-149 G C S 6952 10883 3019 99-21703-36 G A A 6953 10884 3020 99-21705-306 T G A 6954 10885 3021 99-21707-429 C T S 6955 10886 3022 99-21710-272 C G S 6956 10887 3023 99-21733-323 G C S 6957 10888 3024 99-21734-183 C T S 6958 10889 3025 99-21742-337 G A A 6959 10890 3026 99-21745-455 T C S 6960 10891 3027 99-21756-230 T G A 6961 10892 3028 99-21759-21 T G A 6962 10893 3029 99-21762-135 A C S 6963 10894 3030 99-21763-52 A G A 6964 10895 3031 99-21765-111 A T S 6965 10896 3032 99-21767-392 T A S 6966 10897 3033 99-21771-144 G A A 6967 10898 3034 99-21775-466 A T S 6968 10899 3035 99-21787-348 A G A 6969 10900 3036 99-21790-161 G A A 6970 10901 3037 99-21791-364 T C S 6971 10902 3038 99-21800-310 A G A 6972 10903 3039 99-21801-123 T C S 6973 10904 3040 99-21804-310 T C S 6974 10905 3041 99-21810-222 G A A 6975 10906 3042 99-21811-209 T C S 6976 10907 3043 99-21827-155 T C S 6977 10908 3044 99-21829-261 C T S 6978 10909 3045 99-21831-311 A G A 6979 10910 3046 99-21838-153 A G A 6980 10911 3047 99-21844-165 G A A 6981 10912 3048 99-21846-327 C T S 6982 10913 3049 99-21874-311 G T A 6983 10914 3050 99-21880-331 C T S 6984 10915 3051 99-21881-152 T C S 6985 10916 3052 99-21889-219 G A A 6986 10917 3053 99-21893-388 G A A 6987 10918 3054 99-21896-345 A G A 6988 10919 3055 99-21898-102 T A S 6989 10920 3056 99-21901-331 G A A 6990 10921 3057 99-21913-483 A G A 6991 10922 3058 99-21916-359 A G A 6992 10923 3059 99-21917-84 G C S 6993 10924 3060 99-21919-38 A G A 6994 10925 3061 99-21921-338 T C S 6995 10926 3062 99-21943-413 C T S 6996 10927 3063 99-21948-237 C T S 6997 10928 3064 99-21950-107 G C S 6998 10929 3065 99-21952-76 T C A 6999 10930 3066 99-21968-150 G A A 7000 10931 3067 99-21969-425 T G A 7001 10932 3068 99-22008-325 C T S 7002 10933 3069 99-22098-101 C G S 7003 10934 3070 99-22155-199 T C S 7004 10935 3071 99-22181-171 G A A 7005 10936 3072 99-22187-261 C T S 7006 10937 3073 99-22190-369 T C S 7007 10938 3074 99-22202-58 T C S 7008 10939 3075 99-22204-391 T C S 7009 10940 3076 99-22206-455 C G S 7010 10941 3077 99-22213-333 T A S 7011 10942 3078 99-22355-213 T A S 7012 10943 3079 99-22363-268 A G A 7013 10944 3080 99-22375-353 G A A 7014 10945 3081 99-22405-335 C T S 7015 10946 3082 99-2251-151 G A A 7016 10947 3083 99-22530-48 C T S 7017 10948 3084 99-22537-280 A G A 7018 10949 3085 99-22567-243 C G S 7019 10950 3086 99-22572-72 A G A 7020 10951 3087 99-22593-64 C T S 7021 10952 3088 99-22706-367 T C S 7022 10953 3089 99-22729-352 T A S 7023 10954 3090 99-22768-113 G T A 7024 10955 3091 99-22814-349 C T S 7025 10956 3092 99-22818-33 C T S 7026 10957 3093 99-22826-311 C T S 7027 10958 3094 99-22851-121 A G A 7028 10959 3095 99-23113-388 C T S 7029 10960 3096 99-23188-227 T C S 7030 10961 3097 99-23240-326 C A S 7031 10962 3098 99-23246-66 A G A 7032 10963 3099 99-23248-308 A G A 7033 10964 3100 99-23249-262 A G A 7034 10965 3101 99-23274-182 C T S 7035 10966 3102 99-2333-423 T G A 7036 10967 3103 99-2341-485 C T S 7037 10968 3104 99-2342-217 C T S 7038 10969 3105 99-23427-283 G A A 7039 10970 3106 99-23442-190 T C S 7040 10971 3107 99-23544-340 C A S 7041 10972 3108 99-23549-78 G A A 7042 10973 3109 99-23558-98 A G A 7043 10974 3110 99-23565-252 G C S 7044 10975 3111 99-23589-198 A C S 7045 10976 3112 99-23590-205 C T S 7046 10977 3113 99-23621-189 G A A 7047 10978 3114 99-23641-159 G A A 7048 10979 3115 99-23652-244 G A A 7049 10980 3116 99-23696-164 C T S 7050 10981 3117 99-23701-104 C T S 7051 10982 3118 99-23702-437 G A A 7052 10983 3119 99-2371-93 A C S 7053 10984 3120 99-23711-455 C T S 7054 10985 3121 99-23730-202 T C S 7055 10986 3122 99-23736-314 G A S 7056 10987 3123 99-23813-476 C T S 7057 10988 3124 99-23821-176 G C S 7058 10989 3125 99-23844-382 A C S 7059 10990 3126 99-23858-51 G T A 7060 10991 3127 99-23860-146 G C S 7061 10992 3128 99-23876-265 A C S 7062 10993 3129 99-23878-400 C A S 7063 10994 3130 99-23880-268 G A A 7064 10995 3131 99-23887-103 G A A 7065 10996 3132 99-23889-342 A G A 7066 10997 3133 99-23894-339 T C S 7067 10998 3134 99-23895-40 T C S 7068 10999 3135 99-23902-103 T C S 7069 11000 3136 99-23912-116 G C S 7070 11001 3137 99-23915-69 A G A 7071 11002 3138 99-23918-179 T C S 7072 11003 3139 99-23934-353 G A A 7073 11004 3140 99-23936-216 T C S 7074 11005 3141 99-23938-414 G A A 7075 11006 3142 99-23943-245 A G A 7076 11007 3143 99-23960-298 T C A 7077 11008 3144 99-23965-360 C T A 7078 11009 3145 99-23977-141 G A S 7079 11010 3146 99-23987-115 C T A 7080 11011 3147 99-23988-441 C T A 7081 11012 3148 99-23995-407 A G S 7082 11013 3149 99-24000-316 C T S 7083 11014 3150 99-24003-172 T C A 7084 11015 3151 99-24004-200 A G S 7085 11016 3152 99-24007-362 T C A 7086 11017 3153 99-24020-379 C A S 7087 11018 3154 99-24038-103 C T A 7088 11019 3155 99-24063-363 T C S 7089 11020 3156 99-24073-384 G A A 7090 11021 3157 99-24075-45 T G A 7091 11022 3158 99-24079-268 C A S 7092 11023 3159 99-24084-110 G A A 7093 11024 3160 99-24092-209 A G A 7094 11025 3161 99-24096-386 T A S 7095 11026 3162 99-24105-247 C T S 7096 11027 3163 99-24113-332 A G A 7097 11028 3164 99-24117-169 A G A 7098 11029 3165 99-24119-368 T G A 7099 11030 3166 99-24123-125 A G A 7100 11031 3167 99-24140-394 G A A 7101 11032 3168 99-24148-332 A C S 7102 11033 3169 99-24152-268 C T S 7103 11034 3170 99-24155-271 A C S 7104 11035 3171 99-24156-107 C T S 7105 11036 3172 99-24167-85 A C S 7106 11037 3173 99-24175-218 A G A 7107 11038 3174 99-24180-390 A G A 7108 11039 3175 99-24182-326 C A S 7109 11040 3176 99-24185-446 T C S 7110 11041 3177 99-24187-142 A G A 7111 11042 3178 99-24190-231 G C S 7112 11043 3179 99-24202-433 C G S 7113 11044 3180 99-24204-486 T C S 7114 11045 3181 99-24208-292 T A S 7115 11046 3182 99-24210-111 G A A 7116 11047 3183 99-24217-206 T C S 7117 11048 3184 99-24225-439 A G A 7118 11049 3185 99-24228-386 G C S 7119 11050 3186 99-24232-419 A G A 7120 11051 3187 99-24234-352 A G S 7121 11052 3188 99-24369-263 G C S 7122 11053 3189 99-24397-315 G C S 7123 11054 3190 99-24408-202 A G S 7124 11055 3191 99-2441-512 A G A 7125 11056 3192 99-24412-279 C T A 7126 11057 3193 99-24415-85 T C A 7127 11058 3194 99-24470-168 G A S 7128 11059 3195 99-24472-179 A G S 7129 11060 3196 99-24480-44 A C S 7130 11061 3197 99-24485-55 G T A 7131 11062 3198 99-24490-363 A G S 7132 11063 3199 99-24492-351 C G S 7133 11064 3200 99-24581-253 G A S 7134 11065 3201 99-24591-33 T C A 7135 11066 3202 99-24592-55 C T A 7136 11067 3203 99-24745-413 T C A 7137 11068 3204 99-24753-182 G A S 7138 11069 3205 99-24768-233 C T S 7139 11070 3206 99-24855-180 C T A 7140 11071 3207 99-24863-199 C T A 7141 11072 3208 99-24867-219 G A S 7142 11073 3209 99-24871-435 A G S 7143 11074 3210 99-24889-311 T A S 7144 11075 3211 99-24897-276 C G S 7145 11076 3212 99-24904-187 T C A 7146 11077 3213 99-24909-440 A G S 7147 11078 3214 99-24917-250 G A S 7148 11079 3215 99-24930-299 A G A 7149 11080 3216 99-24936-332 G T A 7150 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G A A 7209 11140 3276 99-2938-83 T C S 7210 11141 3277 99-2943-230 T G A 7211 11142 3278 99-2944-351 C T S 7212 11143 3279 99-295-355 T C S 7213 11144 3280 99-2954-160 C G S 7214 11145 3281 99-2956-239 C T S 7215 11146 3282 99-2970-318 G C S 7216 11147 3283 99-2978-135 C A S 7217 11148 3284 99-2981-53 T C S 7218 11149 3285 99-2988-243 C T S 7219 11150 3286 99-2989-345 C A S 7220 11151 3287 99-2991-256 G A A 7221 11152 3288 99-2995-168 C T S 7222 11153 3289 99-2999-371 C T S 7223 11154 3290 99-3013-250 A T S 7224 11155 3291 99-3018-50 A G A 7225 11156 3292 99-3019-316 A T S 7226 11157 3293 99-3020-369 A G A 7227 11158 3294 99-3021-290 A G A 7228 11159 3295 99-3044-216 C T S 7229 11160 3296 99-3045-108 C T S 7230 11161 3297 99-3046-91 T C S 7231 11162 3298 99-3047-395 G A A 7232 11163 3299 99-3058-420 T A S 7233 11164 3300 99-306-119 G A A 7234 11165 3301 99-3061-369 A G A 7235 11166 3302 99-3106-272 G A A 7236 11167 3303 99-3108-156 A T S 7237 11168 3304 99-3109-402 G A A 7238 11169 3305 99-3110-321 C T S 7239 11170 3306 99-312-311 C T S 7240 11171 3307 99-3129-113 T A S 7241 11172 3308 99-3132-158 A G A 7242 11173 3309 99-3144-112 A G A 7243 11174 3310 99-3147-24 C G S 7244 11175 3311 99-3153-190 C T S 7245 11176 3312 99-3154-110 T C S 7246 11177 3313 99-3156-251 T C S 7247 11178 3314 99-3167-227 A G A 7248 11179 3315 99-3195-71 G A A 7249 11180 3316 99-3217-274 G A A 7250 11181 3317 99-3224-232 A G A 7251 11182 3318 99-3231-109 T A S 7252 11183 3319 99-3234-274 A C S 7253 11184 3320 99-325-226 A C S 7254 11185 3321 99-3266-193 G A A 7255 11186 3322 99-3276-195 C A S 7256 11187 3323 99-3279-337 T C S 7257 11188 3324 99-3293-300 T G A 7258 11189 3325 99-3296-101 T A S 7259 11190 3326 99-3299-211 C T S 7260 11191 3327 99-3305-272 A C S 7261 11192 3328 99-3335-53 C T S 7262 11193 3329 99-3337-294 C T S 7263 11194 3330 99-3342-103 G A A 7264 11195 3331 99-3347-226 T A S 7265 11196 3332 99-3349-124 A C S 7266 11197 3333 99-3353-350 T C S 7267 11198 3334 99-3356-345 A G A 7268 11199 3335 99-3368-277 C T S 7269 11200 3336 99-3373-253 C G S 7270 11201 3337 99-3374-274 G A A 7271 11202 3338 99-3385-197 C T S 7272 11203 3339 99-3390-328 G A A 7273 11204 3340 99-3391-160 C T S 7274 11205 3341 99-3393-245 A G A 7275 11206 3342 99-3398-196 T C S 7276 11207 3343 99-3399-449 C T S 7277 11208 3344 99-3400-83 G A A 7278 11209 3345 99-3414-112 G A A 7279 11210 3346 99-3415-215 G A A 7280 11211 3347 99-3426-270 C T S 7281 11212 3348 99-3428-366 A G A 7282 11213 3349 99-3445-239 G C S 7283 11214 3350 99-3453-138 A G A 7284 11215 3351 99-3460-337 C T S 7285 11216 3352 99-3462-117 C T S 7286 11217 3353 99-3468-272 A G A 7287 11218 3354 99-3469-313 C G S 7288 11219 3355 99-3473-309 C G S 7289 11220 3356 99-3474-272 A G S 7290 11221 3357 99-3478-199 G A A 7291 11222 3358 99-3479-293 T C S 7292 11223 3359 99-3482-225 A G A 7293 11224 3360 99-3483-252 T C S 7294 11225 3361 99-3485-245 T A S 7295 11226 3362 99-3511-130 G A A 7296 11227 3363 99-3519-374 G A A 7297 11228 3364 99-3522-210 A G A 7298 11229 3365 99-3523-270 A C S 7299 11230 3366 99-3524-403 T A S 7300 11231 3367 99-3542-336 G T A 7301 11232 3368 99-3556-129 T G A 7302 11233 3369 99-3563-121 C T S 7303 11234 3370 99-3580-122 C G S 7304 11235 3371 99-3588-188 T A S 7305 11236 3372 99-3589-203 C T S 7306 11237 3373 99-3596-147 C T S 7307 11238 3374 99-36-69 C T S 7308 11239 3375 99-3601-226 T C S 7309 11240 3376 99-3603-80 T A S 7310 11241 3377 99-3604-91 A G A 7311 11242 3378 99-3619-330 C T S 7312 11243 3379 99-3620-314 G A A 7313 11244 3380 99-3628-31 G A A 7314 11245 3381 99-3629-219 G A A 7315 11246 3382 99-3631-159 C T S 7316 11247 3383 99-3638-259 A C S 7317 11248 3384 99-3641-230 C A S 7318 11249 3385 99-3666-280 G A A 7319 11250 3386 99-3667-190 G A A 7320 11251 3387 99-3677-196 T A S 7321 11252 3388 99-3680-274 C G S 7322 11253 3389 99-3689-50 A G A 7323 11254 3390 99-3690-355 G C S 7324 11255 3391 99-3699-230 G A A 7325 11256 3392 99-3702-226 T A S 7326 11257 3393 99-3703-331 C T S 7327 11258 3394 99-3705-195 G A A 7328 11259 3395 99-3709-366 T C S 7329 11260 3396 99-3717-68 A G A 7330 11261 3397 99-3728-341 T C S 7331 11262 3398 99-3739-215 G A A 7332 11263 3399 99-3746-337 C G S 7333 11264 3400 99-3749-174 C T S 7334 11265 3401 99-3752-210 C T S 7335 11266 3402 99-3760-59 A G A 7336 11267 3403 99-3761-329 C T S 7337 11268 3404 99-3764-198 C T S 7338 11269 3405 99-3765-279 A G A 7339 11270 3406 99-377-306 G A S 7340 11271 3407 99-3773-337 T C S 7341 11272 3408 99-3774-351 A G A 7342 11273 3409 99-3775-98 G A A 7343 11274 3410 99-3778-97 T A S 7344 11275 3411 99-3789-293 A G A 7345 11276 3412 99-3792-294 A G A 7346 11277 3413 99-3802-197 C T S 7347 11278 3414 99-3805-125 A G A 7348 11279 3415 99-3812-243 T G A 7349 11280 3416 99-3813-122 T C S 7350 11281 3417 99-3857-261 A G A 7351 11282 3418 99-3862-153 A G A 7352 11283 3419 99-3875-138 A C S 7353 11284 3420 99-3888-309 G A A 7354 11285 3421 99-3893-108 A C S 7355 11286 3422 99-3941-107 A G A 7356 11287 3423 99-3944-247 G T A 7357 11288 3424 99-3953-77 G A A 7358 11289 3425 99-3954-362 G C S 7359 11290 3426 99-3978-146 C T S 7360 11291 3427 99-3981-156 A G A 7361 11292 3428 99-3992-185 C T S 7362 11293 3429 99-4001-330 C T S 7363 11294 3430 99-4009-232 C T S 7364 11295 3431 99-4025-300 C T S 7365 11296 3432 99-4052-415 G T A 7366 11297 3433 99-4064-346 A C S 7367 11298 3434 99-4065-20 A G A 7368 11299 3435 99-4073-307 C A S 7369 11300 3436 99-4076-255 G A A 7370 11301 3437 99-4077-230 T C S 7371 11302 3438 99-4078-212 G C S 7372 11303 3439 99-4079-389 A G A 7373 11304 3440 99-4119-307 C T S 7374 11305 3441 99-4120-253 C T S 7375 11306 3442 99-4122-23 T C S 7376 11307 3443 99-4125-192 C A S 7377 11308 3444 99-4131-288 T C S 7378 11309 3445 99-4138-360 A C S 7379 11310 3446 99-4139-128 C T S 7380 11311 3447 99-4140-254 C T S 7381 11312 3448 99-4182-113 A G A 7382 11313 3449 99-4193-384 A G A 7383 11314 3450 99-4194-336 T C S 7384 11315 3451 99-4199-339 G A A 7385 11316 3452 99-4201-501 G T A 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99-4398-167 T A S 7416 11347 3483 99-4399-228 T A S 7417 11348 3484 99-4404-384 G A A 7418 11349 3485 99-4406-115 A G A 7419 11350 3486 99-4435-203 A G A 7420 11351 3487 99-4448-174 T C S 7421 11352 3488 99-4455-357 T A S 7422 11353 3489 99-4458-59 A G A 7423 11354 3490 99-4467-39 T C S 7424 11355 3491 99-4468-130 C A S 7425 11356 3492 99-4483-333 C T S 7426 11357 3493 99-4534-158 T C S 7427 11358 3494 99-4567-424 T C A 7428 11359 3495 99-4575-226 C T S 7429 11360 3496 99-4580-296 G A A 7430 11361 3497 99-4589-169 C T S 7431 11362 3498 99-4614-72 A C S 7432 11363 3499 99-4619-267 A C S 7433 11364 3500 99-4636-62 C T S 7434 11365 3501 994649-251 T A S 7435 11366 3502 99-468-271 T C S 7436 11367 3503 99-4688-442 C G S 7437 11368 3504 99-4691-400 A G A 7438 11369 3505 99-4692-372 T G A 7439 11370 3506 99-4715-280 G A A 7440 11371 3507 99-4736-164 C T S 7441 11372 3508 99-4744-72 G T A 7442 11373 3509 99-4746-160 G T A 7443 11374 3510 99-4748-76 C T S 7444 11375 3511 99-4755-84 C A S 7445 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G A 7475 11406 3542 99-5347-394 T C S 7476 11407 3543 99-5397-353 G C A 7477 11408 3544 99-55-233 C A S 7478 11409 3545 99-5549-289 A G A 7479 11410 3546 99-5569-237 A G A 7480 11411 3547 99-5575-330 C T S 7481 11412 3548 99-5602-372 G C S 7482 11413 3549 99-5671-333 T C S 7483 11414 3550 99-568-101 G A S 7484 11415 3551 99-5688-116 A G A 7485 11416 3552 99-5689-391 G C S 7486 11417 3553 99-5715-224 T G A 7487 11418 3554 99-5718-82 A G A 7488 11419 3555 99-5723-291 A G A 7489 11420 3556 99-5747-278 T C S 7490 11421 3557 99-5775-154 C T S 7491 11422 3558 99-5828-235 T C S 7492 11423 3559 99-5846-383 C T S 7493 11424 3560 99-5861-151 G A A 7494 11425 3561 99-59-137 A C S 7495 11426 3562 99-5930-449 T A S 7496 11427 3563 99-5931-330 G A A 7497 11428 3564 99-5967-165 C T S 7498 11429 3565 99-5987-135 A G A 7499 11430 3566 99-5996-279 T C S 7500 11431 3567 99-6001-372 T G A 7501 11432 3568 99-6020-477 A G A 7502 11433 3569 99-6047-225 T A S 7503 11434 3570 99-6071-272 A G A 7504 11435 3571 99-6076-394 A T S 7505 11436 3572 99-6096-354 C G S 7506 11437 3573 99-6103-356 T C S 7507 11438 3574 99-6124-125 T C S 7508 11439 3575 99-6173-229 T G A 7509 11440 3576 99-634-278 T A A 7510 11441 3577 99-6401-64 A G A 7511 11442 3578 99-6538-193 G A A 7512 11443 3579 99-6549-275 A G A 7513 11444 3580 99-6564-236 G C S 7514 11445 3581 99-6574-150 T G A 7515 11446 3582 99-6583-289 A G A 7516 11447 3583 99-6591-236 T C S 7517 11448 3584 99-6597-213 A G A 7518 11449 3585 99-6603-47 A G A 7519 11450 3586 99-6707-405 G A A 7520 11451 3587 99-6720-186 C G S 7521 11452 3588 99-6809-317 C T S 7522 11453 3589 99-6834-307 G A A 7523 11454 3590 99-6837-253 C A S 7524 11455 3591 99-6878-317 C T S 7525 11456 3592 99-6888-188 T G A 7526 11457 3593 99-6919-372 G A A 7527 11458 3594 99-6922-169 T C S 7528 11459 3595 99-6974-417 A G A 7529 11460 3596 99-6978-149 C G S 7530 11461 3597 99-6984-287 G C S 7531 11462 3598 99-6998-86 T C S 7532 11463 3599 99-7032-416 T A S 7533 11464 3600 99-7048-342 C T S 7534 11465 3601 99-7060-512 A G A 7535 11466 3602 99-7086-91 G C S 7536 11467 3603 99-7117-266 T A S 7537 11468 3604 99-7203-286 T C A 7538 11469 3605 99-7268-383 T C S 7539 11470 3606 99-7281-131 T C A 7540 11471 3607 99-7282-145 G T A 7541 11472 3608 99-7296-429 T C S 7542 11473 3609 99-7344-203 C T S 7543 11474 3610 99-7377-370 T C S 7544 11475 3611 99-7394-398 A G A 7545 11476 3612 99-7412-288 C T S 7546 11477 3613 99-7422-375 C T S 7547 11478 3614 99-7430-548 T A S 7548 11479 3615 99-7442-390 T C S 7549 11480 3616 99-7481-268 A G A 7550 11481 3617 99-7696-215 C T S 7551 11482 3618 99-7702-225 C T S 7552 11483 3619 99-7772-185 C T S 7553 11484 3620 99-7815-70 A T S 7554 11485 3621 99-7818-342 G A A 7555 11486 3622 99-7860-320 T G A 7556 11487 3623 99-7886-350 G C S 7557 11488 3624 99-7944-130 A T S 7558 11489 3625 99-7945-106 G A A 7559 11490 3626 99-7976-324 A G A 7560 11491 3627 99-8000-88 C T S 7561 11492 3628 99-8006-241 C T S 7562 11493 3629 99-8038-47 T C S 7563 11494 3630 99-8055-299 A G A 7564 11495 3631 99-8059-59 A G A 7565 11496 3632 99-8061-106 C T S 7566 11497 3633 99-8109-168 A G A 7567 11498 3634 99-8115-238 T G A 7568 11499 3635 99-8166-370 T C S 7569 11500 3636 99-8226-78 T G A 7570 11501 3637 99-8232-303 A G A 7571 11502 3638 99-824-359 C T S 7572 11503 3639 99-8274-70 A G A 7573 11504 3640 99-8359-153 T A S 7574 11505 3641 99-8630-298 G A A 7575 11506 3642 99-8659-399 T A S 7576 11507 3643 99-8679-371 T G A 7577 11508 3644 99-8690-117 T C S 7578 11509 3645 99-8751-299 C T S 7579 11510 3646 99-8795-58 G A A 7580 11511 3647 99-882-250 C A S 7581 11512 3648 99-887-344 G T A 7582 11513 3649 99-8875-283 G A A 7583 11514 3650 99-892-77 T C S 7584 11515 3651 99-8936-202 T C S 7585 11516 3652 99-8952-319 G C S 7586 11517 3653 99-896-83 T C S 7587 11518 3654 99-899-252 G C S 7588 11519 3655 99-9072-32 G C S 7589 11520 3656 99-9076-357 C T S 7590 11521 3657 99-9077-52 T C S 7591 11522 3658 99-9089-155 T A S 7592 11523 3659 99-9113-277 C A S 7593 11524 3660 99-9145-438 A G A 7594 11525 3661 99-9164-365 T C S 7595 11526 3662 99-9308-416 A G A 7596 11527 3663 99-9316-399 A G A 7597 11528 3664 99-9343-71 C T S 7598 11529 3665 99-9362-282 T C S 7599 11530 3666 99-9363-143 G A A 7600 11531 3667 99-9375-337 G C S 7601 11532 3668 99-9380-292 T C S 7602 11533 3669 99-9607-402 C T A 7603 11534 3670 99-9620-241 C T S 7604 11535 3671 99-9623-330 T C S 7605 11536 3672 99-9633-32 T C S 7606 11537 3673 99-9636-423 T C S 7607 11538 3674 99-9658-42 T G A 7608 11539 3675 99-9662-213 T C A 7609 11540 3676 99-9666-363 T C A 7610 11541 3677 99-9668-185 C T A 7611 11542 3678 99-9680-363 A G S 7612 11543 3679 99-9696-292 T C A 7613 11544 3680 99-9697-375 A C S 7614 11545 3681 99-9700-289 C T A 7615 11546 3682 99-9704-445 T C A 7616 11547 3683 99-9706-448 C T A 7617 11548 3684 99-9709-115 T C A 7618 11549 3685 99-9710-242 C A S 7619 11550 3686 99-9714-302 T C S 7620 11551 3687 99-9717-449 A G A 7621 11552 3688 99-9726-190 T C S 7622 11553 3689 99-974-231 A G A 7623 11554 3690 99-9745-284 G A A 7624 11555 3691 99-9751-134 A T S 7625 11556 3692 99-9765-237 A G S 7626 11557 3693 99-9774-392 A T S 7627 11558 3694 99-9778-360 A G S 7628 11559 3695 99-9781-174 T C A 7629 11560 3696 99-9785-141 T C A 7630 11561 3697 99-9810-257 A T S 7631 11562 3698 99-9811-369 C T A 7632 11563 3699 99-9820-483 C T A 7633 11564 3700 99-9822-257 A T S 7634 11565 3701 99-9829-367 G A S 7635 11566 3702 99-983-278 G A A 7636 11567 3703 99-9832-128 T C A 7637 11568 3704 99-9833-167 A G S 7638 11569 3705 99-9835-217 C T A 7639 11570 3706 99-9837-275 A G S 7640 11571 3707 99-9839-416 G C S 7641 11572 3708 99-9840-192 C T A 7642 11573 3709 99-9847-25 A G S 7643 11574 3710 99-9849-291 G A S 7644 11575 3711 99-9852-276 T C A 7645 11576 3712 99-9854-316 G C S 7646 11577 3713 99-9856-252 C T A 7647 11578 3714 99-9859-132 C A S 7648 11579 3715 99-9866-365 T C A 7649 11580 3716 99-990-356 T A S 7650 11581 3717 99-9906-280 G C S 7651 11582 3718 99-9908-423 T A S 7652 11583 3719 99-991-157 A G A 7653 11584 3720 99-9915-281 T G A 7654 11585 3721 99-9920-245 T C A 7655 11586 3722 99-9921-365 T C A 7656 11587 3723 99-9922-154 C T A 7657 11588 3724 99-9926-454 G A S 7658 11589 3725 99-9928-454 C T A 7659 11590 3726 99-9929-144 G A S 7660 11591 3727 99-9935-418 G A S 7661 11592 3728 99-9941-426 T A S 7662 11593 3729 99-995-251 A C S 7663 11594 3730 99-996-210 T C S 7664 11595 3731 99-9986-202 T C S 7665 11596 3732 99-9988-111 T G A 7666 11597 3733 99-9994-226 C A S 7667 11598 3734 99-9995-50 C T S 7668 11599 3735 99-10069-366 A T S 7669 11600 3736 99-10074-266 A G A 7670 11601 3737 99-10129-177 A G S 7671 11602 3738 99-10198-271 A G S 7672 11603 3739 99-10306-345 C T A 7673 11604 3740 99-10307-115 A G S 7674 11605 3741 99-10326-149 C T A 7675 11606 3742 99-10393-179 A G S 7676 11607 3743 99-10685-454 A C S 7677 11608 3744 99-10857-217 C T A 7678 11609 3745 99-10948-281 C T A 7679 11610 3746 99-11104-329 A G S 7680 11611 3747 99-11116-199 C T A 7681 11612 3748 99-11117-282 A G S 7682 11613 3749 99-11121-461 A G S 7683 11614 3750 99-11124-363 C T A 7684 11615 3751 99-11172-373 C T A 7685 11616 3752 99-11206-379 C T A 7686 11617 3753 99-11303-223 C T A 7687 11618 3754 99-11307-168 G T A 7688 11619 3755 99-11325-188 A G S 7689 11620 3756 99-11365-273 C T A 7690 11621 3757 99-11389-268 A T S 7691 11622 3758 99-11395-376 A G S 7692 11623 3759 99-11500-50 C T S 7693 11624 3760 99-11571-88 G T S 7694 11625 3761 99-11710-452 A G S 7695 11626 3762 99-1173-208 A T S 7696 11627 3763 99-11735-215 C T A 7697 11628 3764 99-11864-218 A C S 7698 11629 3765 99-1187-293 G C S 7699 11630 3766 99-11872-228 C T A 7700 11631 3767 99-11878-212 C T A 7701 11632 3768 99-11905-202 G C S 7702 11633 3769 99-11932-48 C T A 7703 11634 3770 99-11964-158 A C S 7704 11635 3771 99-12164-412 C T A 7705 11636 3772 99-12227-278 G C S 7706 11637 3773 99-12417-447 A G S 7707 11638 3774 99-12459-119 G T A 7708 11639 3775 99-12521-212 C T S 7709 11640 3776 99-12569-95 A G A 7710 11641 3777 99-1298-430 A G S 7711 11642 3778 99-1315-105 G C S 7712 11643 3779 99-13154-74 C T A 7713 11644 3780 99-13155-134 A G S 7714 11645 3781 99-13249-461 C T A 7715 11646 3782 99-13794-147 G C S 7716 11647 3783 99-14899-215 A C S 7717 11648 3784 99-16351-44 C T A 7718 11649 3785 99-16436-382 A G S 7719 11650 3786 99-16753-387 G C S 7720 11651 3787 99-1807-300 A G A 7721 11652 3788 99-19032-132 A C S 7722 11653 3789 99-19212-369 C T S 7723 11654 3790 99-19273-219 A G A 7724 11655 3791 99-19279-356 C T S 7725 11656 3792 99-19541-172 A G A 7726 11657 3793 99-19552-214 G T A 7727 11658 3794 99-21051-435 C T S 7728 11659 3795 99-21246-20 C T S 7729 11660 3796 99-21387-465 C T S 7730 11661 3797 99-21407-352 A G A 7731 11662 3798 99-21418-83 C T S 7732 11663 3799 99-21419-85 C T S 7733 11664 3800 99-21430-308 C T S 7734 11665 3801 99-21435-96 A G A 7735 11666 3802 99-21446-240 C T S 7736 11667 3803 99-21452-173 A G A 7737 11668 3804 99-21488-376 G T A 7738 11669 3805 99-21489-227 C T S 7739 11670 3806 99-21496-248 C T S 7740 11671 3807 99-21519-446 A G A 7741 11672 3808 99-21618-178 A G A 7742 11673 3809 99-21725-371 C T S 7743 11674 3810 99-21773-155 A C S 7744 11675 3811 99-21781-252 A G A 7745 11676 3812 99-21820-230 A G A 7746 11677 3813 99-21822-50 A G A 7747 11678 3814 99-21939-170 A T S 7748 11679 3815 99-22404-59 A G A 7749 11680 3816 99-22594-395 A G A 7750 11681 3817 99-22679-148 A C S 7751 11682 3818 99-23095-184 G C S 7752 11683 3819 99-23370-249 C T S 7753 11684 3820 99-23568-395 G C S 7754 11685 3821 99-23824-339 C T S 7755 11686 3822 99-23969-316 C T A 7756 11687 3823 99-24032-138 A T S 7757 11688 3824 99-24048-286 C T S 7758 11689 3825 99-24074-190 A G A 7759 11690 3826 99-24082-408 A C S 7760 11691 3827 99-24104-308 G T A 7761 11692 3828 99-24138-224 A G A 7762 11693 3829 99-24172-116 C T S 7763 11694 3830 99-24267-190 A C S 7764 11695 3831 99-24949-289 C T A 7765 11696 3832 99-253-97 A G A 7766 11697 3833 99-2694-411 A G A 7767 11698 3834 99-2697-336 A G A 7768 11699 3835 99-2720-280 A G A 7769 11700 3836 99-2851-105 G C S 7770 11701 3837 99-2889-197 C T S 7771 11702 3838 99-3072-323 A G A 7772 11703 3839 99-3089-49 A G A 7773 11704 3840 99-3157-203 G T A 7774 11705 3841 99-3210-341 G T A 7775 11706 3842 99-3218-344 A G A 7776 11707 3843 99-3251-254 G T A 7777 11708 3844 99-3298-158 C T A 7778 11709 3845 99-3300-433 A G A 7779 11710 3846 99-3364-247 A T S 7780 11711 3847 99-3427-271 A G A 7781 11712 3848 99-3484-96 A G A 7782 11713 3849 99-3537-196 A G A 7783 11714 3850 99-3568-156 G T A 7784 11715 3851 99-3592-325 A G A 7785 11716 3852 99-3602-245 C T S 7786 11717 3853 99-3608-264 A G A 7787 11718 3854 99-3643-352 A G A 7788 11719 3855 99-3770-363 C T S 7789 11720 3856 99-3772-266 A G A 7790 11721 3857 99-3790-361 A G A 7791 11722 3858 99-3818-255 A G A 7792 11723 3859 99-3863-328 A G A 7793 11724 3860 99-3879-245 A G A 7794 11725 3861 99-3882-312 C T S 7795 11726 3862 99-3883-329 C T S 7796 11727 3863 99-3884-355 G C S 7797 11728 3864 99-3894-333 C T S 7798 11729 3865 99-3936-352 A G S 7799 11730 3866 99-3946-236 A G A 7800 11731 3867 99-4029-174 C T S 7801 11732 3868 99-4036-308 C T S 7802 11733 3869 99-4102-109 A G A 7803 11734 3870 99-4110-180 A G A 7804 11735 3871 99-4111-259 A G A 7805 11736 3872 99-4126-366 A G A 7806 11737 3873 99-4157-72 A G A 7807 11738 3874 99-4228-168 C T S 7808 11739 3875 99-4239-328 A G A 7809 11740 3876 99-4254-307 A G A 7810 11741 3877 99-4264-228 C T S 7811 11742 3878 99-4311-146 A G A 7812 11743 3879 99-4381-385 C T S 7813 11744 3880 99-4403-194 A C S 7814 11745 3881 99-4524-296 A G A 7815 11746 3882 99-4582-359 G T A 7816 11747 3883 99-4611-151 C T S 7817 11748 3884 99-4689-375 A T S 7818 11749 3885 994762-114 A G A 7819 11750 3886 99-4878-107 C T S 7820 11751 3887 99-5075-219 C T A 7821 11752 3888 99-5190-277 A G A 7822 11753 3889 99-5605-90 G T A 7823 11754 3890 99-5882-105 C T S 7824 11755 3891 99-5977-241 C T S 7825 11756 3892 99-5993-323 C T S 7826 11757 3893 99-5994-205 G T S 7827 11758 3894 99-6827-399 A G A 7828 11759 3895 99-7076-198 C T S 7829 11760 3896 99-7215-279 C T S 7830 11761 3897 99-8206-133 A G A 7831 11762 3898 99-8614-236 A G A 7832 11763 3899 99-889-153 G C S 7833 11764 3900 99-9450-70 A T S 7834 11765 3901 99-9609-220 C T A 7835 11766 3902 99-9612-324 A G S 7836 11767 3903 99-9616-136 A G A 7837 11768 3904 99-9683-49 A G S 7838 11769 3905 99-9907-88 C T A 7839 11770 3906 99-993-218 C T S 7840 11771 3907 99-24069-351 C T S 7841 11772 3908 99-3855-279 G C A 7842 11773 3909 99-344-439 G A A 7843 11774 3910 99-366-274 C T S 7844 11775 3911 99-359-308 A G A 7845 11776 3912 99-355-219 A G A 7846 11777 3913 99-365-344 C T S 7847 11778 3914 99-2452-54 C T S 7848 11779 3915 99-123-381 C T S 7849 11780 3916 4-26-29 A G A 7850 11781 3917 4-14-240 C T S 7851 11782 3918 4-77-151 G C S 7852 11783 3919 99-217-277 C T S 7853 11784 3920 4-67-40 C T S 7854 11785 3921 99-213-164 A G A 7855 11786 3922 99-221-377 A C S 7856 11787 3923 99-135-196 A G A 7857 11788 3924 99-1482-32 A C S 7858 11789 3925 4-73-134 G C S 7859 11790 3926 4-65-324 C T S 7860 11791 3927 10-32-357 A C S 7861 11792 3928 10-33-175 T C S 7862 11793 3929 10-33-234 A C S 7862 11793 3930 10-33-327 C T S 7862 11793 3931 10-35-358 G C A 7863 11794 3932 10-35-390 T C S 7863 11794 3933 10-36-164 A G A 7864 11795 3934 10-204-326 A G A 7865 11796

TABLE 9 SEQ Marker Chromosomal ID No. Name Localization Adjacent STS (including aliases) 2 9-1126-384 10p12.1-p11.2 g12982/WI-15761/EST230735/RH51226/R45505 g26880/SRGC-2047/Z24310 g26882/SHGC-14408 g4194/AFMa109xe1/D10S1641/ 9 9-1217-332 21q11.2-q21 g7903/D21S1880/ 59 9-1263-276 21q21 g7833/D21S177 g7957/D21S409/ 236 9-1367-287 1q43 g401/D1S2483/G04024 g428/EST386335/RH50010/SGC35175/ 238 9-13678-251 1q43 g15623/D1S547/GATA4A09 g17350/RH1290/SHGC-477 g17708/RH11033/D29436/D29436 g18500/RH26479/R65593 g319/EST161941/RH50228/RH64454/SGC33718/T91820 g405/D1S1707/G02394 g408/AFM214xe11/Z66804 g417/D1S2421/WI-9317/D29955/RH49709 g420/WI-15754/RH49799 g426/WI-13731/RH49867/RH64343/R44970/ 239 9-13679-285 1q43 g15623/D1S547/GATA4A09 g17350/RH1290/SHGC-477 g17708/RH11033/D29436/D29436 g18500/RH26479/R65593 g319/EST161941/RH50228/RH64454/SGC33718/T91820 g405/D1S1707/G02394 g408/AFM214xe11/Z66804 g417/D1S2421/WI-9317/D29955/RH49709 g420/WI-15754/RH49799 g426/WI-13731/RH49867/RH64343/R44970/ 240 9-1368-299 1q43 g401/D1S2483/G04024 g428/EST386335/RH50010/SGC35175/ 241 9-13684-488 1q43 g15623/D1S547/GATA4A09 g17350/RH1290/SHGC-477 g17708/RH11033/D29436/D29436 g18500/RH26479/R65593 g319/EST161941/RH50228/RH64454/SGC33718/T91820 g405/D1S1707/G02394 g408/AFM214xe11/Z66804 g417/D1S2421/WI-9317/D29955/RH49709 g420/WI-15754/RH49799 g426/WI-13731/RH49867/RH64343/R44970/ 242 9-13687-316 1q43 g15623/D1S547/GATA4A09 g17350/RH1290/SHGC-477 g17708/RH11033/D29436/D29436 g18500/RH26479/R65593 g319/EST161941/RH50228/RH64454/SGC33718/T91820 g405/D1S1707/G02394 g408/AFM214xe11/Z66804 g417/D1S2421/WI-9317/D29955/RH49709 g420/WI-15754/RH49799 g426/WI-13731/RH49867/RH64343/R44970 243 9-1373-358 1q43 g401/D1S2483/G04024 g428/EST386335/RH50010/SGC35175/ 244 9-1376-196 1q43 g401/D1S2483/G04024 g428/EST386335/RH50010/SGC35175/ 245 9-13790-129 1q43 g1262/AFM088xe5/D1S2850 g17846/RH12368/stSG3536 g18893/RH34172/L18266/SHGC-4752 g307/WI-11654/RH50099/RH63795/R10130 g317/EST382595/RR49984/SGC34592 g402/D1S1680/G09467 g421/WI-12850/RH50542/Z41492 g4882/AFMa245wd5/D1S2678 g4901/AFMa247wg9/D1S2680/ 246 9-13798-284 1q43 g1262/AFM088xe5/D1S2850 g17846/RH12368/stSG3536 g18893/RH34172/L18266/SHGC-4752 g307/WI-11654/RH50099/RH63795/R10130 g317/EST382595/RH49984/SGC34592 g402/D1S1680/G09467 g421/WI-12850/RH50542/Z41492 g4882/AFMa245wd5/D1S2678 g4901/AFMa247wg9/D1S2680/ 250 9-1385-91 1q43 g313/D1S3401/G04332 g404/D1S3450/G11836/ 257 9-1387-462 1q43 g313/D1S3401/G04332 g404/D1S3450/G11836/ 260 9-1388-242 1q43 g313/D1S3401/G04332 g404/D1S3450/G11836/ 267 9-1391-204 1q43 g313/D1S3401/G04332 g404/D1S3450/G11836/ 271 9-1392-200 1q43 g313/D1S3401/G04332 g404/D1S3450/G11836/ 276 9-1394-271 1q43 g313/D1S3401/G04332 g404/D1S3450/G11836/ 304 9-1413-137 1q43;5q34 g410/D1S180/ 311 9-1416-589 1q43;5q34 g410/D1S180/ 328 9-14277-73 1q43 g416/D1S3481/G13394/ 329 9-14282-334 1q43 g416/D1S3481/G13394/ 330 9-14285-381 1q43 g416/D1S3481/G13394/ 331 9-14286-220 1q43 g416/D1S3481/G13394/ 332 9-14309-259 2q35 g1392/AFM119xc7/D25126 g4167/AFMa104yd5/D2S2148/ 333 9-14315-405 2q35 g1392/AFM119xc7/D2S126 g4167/AFMa104yd5/D2S2148/ 334 9-14329-205 2q35 g1392/AFM119xc7/D2S126 g4167/AFMa104yd5/D2S2148/ 335 9-14331-64 2q35 g1392/AFM119xc7/D2S126 g4167/AFMa104yd5/D2S2148/ 336 9-14332-437 2q35 g1392/AFM119xc7/D2S126 g4167/AFMa104yd5/D2S2148/ 340 9-1437-325 1q43 g18377/RH25341/D29955 g425/WI-12765/RH50388/RH63951/Z39518/ 346 9-1442-224 1q43 g18377/RH25341/D29955 g425/WI-12765/RH50388/RH63951/Z39518/ 351 9-14468-247 2q32 g650/RH18030/RH70596/ 352 9-14470-243 2q32 g650/RH18030/RH70596/ 353 9-14492-322 2q33 g1453/AFM135xf12/D2S2396/ 354 9-14497-220 2q33 g1453/AFM135xf12/D2S2396/ 355 9-14505-250 5q31.3 g2885/AFM282wd5/D5S638/ 356 9-14518-57 11p15.5-p15.4 g6052/AFMb355za9/D11S4177/ 357 9-1453-204 4p14 g3928/AFMa061zh9/D4S3040/ 381 9-1462-238 8p23.2-p23.1 g480/WI-9756/G05400/ 409 9-1468-435 8p23.2-p23.1 g480/WI-9756/G05400/ 410 9-1469-47 8p23.2-p23.1 g480/WI-9756/G05400/ 418 9-1471-571 8p23.2-p23.1 g480/WI-9756/G05400/ 423 9-1472-435 8p23.2-p23.1 g480/WI-9756/G05400/ 428 9-1474-156 8p23.2-p23.1 g480/WI-9756/G05400/ 432 9-1476-172 8p23.2-p23.1 g480/WI-9756/G05400/ 494 9-14950-346 5q31.1-q31.2 g1446/AFM127xh4/D5S402/ 495 9-14959-81 1q43 g423/WI-18277/RH49873 g424/WI-11392/RH49759/RH63907/T87504/ 496 9-14961-193 1q43 g423/WI-18277/RH49873 g424/WI-11392/RH49759/RH63907/T87504/ 497 9-14962-120 1q43 g423/WI-18277/RH49873 g424/WI-11392/RH49759/RR63907/T87504/ 498 9-14966-187 1q43 g423/WI-18277/RH49873 g424/WI-11392/RH49759/RH63907/T87504/ 499 9-14970-352 2q33 g3108/AFM297ve9/D252336 g648/EST180027/RH56357/SGC34048/R09731/ 500 9-14978-200 2q33 g3108/AFM297ve9/D2S2336 g648/EST180027/RH56357/SGC34048/R09731/ 502 9-14983-186 2q33 g3108/AFM297ve9/D2S2336 g648/EST180027/RH56357/SGC34048/R09731/ 503 9-14984-35 2q33 g3108/AFM297ve9/D252336 g648/EST180027/RH56357/SGC34048/R09731/ 504 9-15005-169 1q42.3-q43 g427/AFMa111yd5/Z67285/ 505 9-15007-369 1q42.3-q43 g427/AFMa111yd5/Z67285/ 507 9-15016-293 2p24 g2364/AFM234vg5/D25309/ 508 9-15018-270 2p24 g2364/AFM234vg5/D25309/ 509 9-15019-408 2p24 g2364/AFM234vg5/D25309/ 510 9-15021-189 2p24 g2364/AFM234vg5/D25309/ 511 9-15030-271 2q33.3-q34 g24928/SHGC-1643/Z24076 g2972/AFM289vf5/D2S346/ 512 9-15039-277 2q33.3-q34 g24928/SHGC-1643/Z24076 g2972/AFM289vf5/D2S346/ 514 9-15043-175 2q33.3-q34 g24928/SHGC-1643/Z24076 g2972/AFM289vf5/D2S346/ 515 9-15046-54 2q33.3-q34 g24928/SHGC-1643/Z24076 g2972/AFM289vf5/D2S346/ 543 9-15290-343 5q31.3 g1094/AFM042xd12/D5S393/ 544 9-15296-326 5q31.3 g1094/AFM042xd12/D5S393/ 545 9-15302-371 5q31.3 g1094/AFM042xd12/D5S393/ 546 9-15307-251 2q34-q35 g1698/AFM172xg3/D2S137 g19419/RH56366/R00076/SGC33908/RH56366 g885/WI-17547/EST253706/R76848/ 547 9-15310-385 2q34-q35 g1698/AFM172xg3/D2S137 g19419/RH56366/R00076/SGC33908/RH56366 g885/WI-17547/EST253706/R76848/ 548 9-15325-95 2q34-q35 g1698/AFM172xg3/D2S137 g19419/RH56366/R00076/SGC33908/RH56366 g885/WI-17547/EST253706/R76848/ 549 9-15328-328 2q34-q35 g1698/AFM172xg3/D2S137 g19419/RH56366/R00076/SGC33908/RH56366 g885/WI-17547/EST253706/R76848/ 551 9-15330-301 2q34-q35 g1698/AFM172xg3/D2S137 g19419/RH56366/R00076/SGC33908/RH56366 g885/WI-17547/EST253706/R76848/ 552 9-15335-313 1q43 g422/WI-15487/RH50392/RH64322/R39926 g431/WI-31075/RH50186/RH64283/SGC31075/ 553 9-15339-378 1q43 g422/WI-15487/RH50392/RH64322/R39926 g431/WI-31075/RH50186/RH64283/SGC31075/ 554 9-15345-376 11p15.5-p15.4 g6052/AFMb355za9/D11S4177/ 570 9-1549-124 1q43 g1385/AFM116xf8/D1S304 g407/AFM151XB8/Z66679/ 584 9-1553-544 1q43 g1385/AFM116xf8/D1S304 g407/AFM151XB8/Z66679/ 588 9-1557-251 1q43 g1385/AFM116xf8/D1S304 g407/AFM151XB8/Z66679/ 591 9-1558-26 1q43 g1385/AFM116xf8/D1S304 g407/AFM151XB8/Z66679/ 597 9-15625-299 1q43 g17455/AFMa045zc5 g309/WI-14972/RH50726/RH64224 g311/WI-14095/RH50061/RH64366/R55784 g403/WI-7199/RH49904/M30269 g406/AFM093XG5/Z66633/ 598 9-15627-324 1q43 g17455/AFMa045zc5 g309/WI-14972/RH50726/RH64224 g311/WI-14095/RH50061/RH64366/R55784 g403/WI-7199/RH49904/M30269 g406/AFM093XG5/Z66633/ 599 9-15636-159 1q43 g17455/AFMa045zc5 g309/WI-14972/RH50726/RH64224 g311/WI-14095/RH50061/RH64366/R55784 g403/WI-7199/RH49904/M30269 g406/AFM093XG5/Z66633/ 600 9-15638-65 Xp11.22-p11.21 g4806/AFMa230vc1/DXS8032/ 601 9-15648-83 Xp11.22-p11.21 g4806/AFMa230vc1/DXS8032/ 602 9-15659-332 Xp21.3-p21.2 g3025/AFM292wb9/DXS1218/ 603 9-1568-240 1q43 g1385/AFM116xf8/D1S304 g407/AFM151XB8/Z66679/ 607 9-1572-440 1q43 g1262/AFM088xe5/D1S2850 g17846/RH12368/stSG3536 g18893/RH34172/L18266/SHGC-4752 g307/WI-11654/RH50099/RH63795/R10130 g317/EST382595/RH49984/SGC34592 g402/D1S1680/G09467 g421/WI-12850/RH50542/Z41492 g4882/AFMa245wd5/D1S2678 g4901/AFMa247wg9/D1S2680/ 616 9-1577-105 1q43 g1262/AFM088xe5/D1S2850 g17846/RH12368/stSG3536 g18893/RH34172/L18266/SHGC-4752 g307/WI-11654/RH50099/RH63795/R10130 g317/EST382595/RH49984/SGC34592 g402/D1S1680/G09467 g421/WI-12850/RH50542/Z41492 g4882/AFMa245wd5/D1S2678 g4901/AFMa247wg9/D1S2680/ 619 9-1578-496 1q43 g1262/AFM088xe5/D1S2850 g17846/RH12368/stSG3536 g18893/RH34172/L18266/SHGC-4752 g307/WI-11654/RH50099/RH63795/R10130 g317/EST382595/RH49984/SGC34592 g402/D1S1680/G09467 g421/WI-12850/RH50542/Z41492 g4882/AFMa245wd5/D1S2678 g4901/AFMa247wg9/D1S2680/ 623 9-1582-430 1q43 g1262/AFM088xe5/D1S2850 g17846/RH12368/stSG3536 g18893/RH34172/L18266/SHGC-4752 g307/WI-11654/RH50099/RH63795/R10130 g317/EST382595/RH49984/SGC34592 g402/D1S1680/G09467 g421/WI-12850/RH50542/Z41492 g4882/AFMa245wd5/D1S2678 g4901/AFMa247wg9/D1S2680/ 626 9-1585-373 1q43 g1262/AFM088xe5/D1S2850 g17846/RH12368/stSG3536 g18893/RH34172/L18266/SHGC-4752 g307/WI-11654/RH50099/RH63795/R10130 g317/EST382595/RH49984/SGC34592 g402/D1S1680/G09467 g421/WI-12850/RH50542/Z41492 g4882/AFMa245wd5/D1S2678 g4901/AFMa247wg9/D1S2680/ 627 9-1587-281 1q43 g1262/AFM088xe5/D1S2850 g17846/RH12368/stSG3536 g18893/RH34172/L18266/SHGC-4752 g307/WI-11654/RH50099/RH63795/R10130 g317/EST382595/RH49984/SGC34592 g402/D1S1680/G09467 g421/WI-12850/RH50542/Z41492 g4882/AFMa245wd5/D1S2678 g4901/AFMa247wg9/D1S2680/ 629 9-1590-116 16q13;2q21 g12752/WI-15065/EST284024/RH54840 g15308/D16S2966/UTR-01731 g23548/RH54739/R50764/WI-14274/ 630 9-15916-270 16q13;2q21 g12752/WI-15065/EST284024/RH54840 g15308/D16S2966/UTR-01731 g23548/RH54739/R50764/WI-14274/ 631 9-15925-331 16q13;2q21 g12752/WI-15065/EST284024/RH54840 g15308/D16S2966/UTR-01731 g23548/RH54739/R50764/WI-14274/ 632 9-15947-109 16q13;2q21 g12752/WI-15065/EST284024/RH54840 g15308/D16S2966/UTR-01731 g23548/RH54739/R50764/WI-14274/ 636 9-1597-162 1q43 g1262/AFM088xe5/D1S2850 g17846/RH12368/stSG3536 g18893/RH34172/L18266/SHGC-4752 g307/WI-11654/RH50099/RH63795/R10130 g317/EST382595/RH49984/SGC34592 g402/D1S1680/G09467 g421/WI-12850/RH50542/Z41492 g4882/AFMa245wd5/D1S2678 g4901/AFMa247wg9/D1S2680/ 645 9-1601-402 1q43 g1262/AFM088xe5/D1S2850 g17846/RH12368/stSG3536 g18893/RH34172/L18266/SHGC-4752 g307/WI-11654/RH50099/RH63795/R10130 g317/EST382595/RH49984/SGC34592 g402/D1S1680/G09467 g421/WI-12850/RH50542/Z41492 g4882/AFMa24Swd5/D1S2678 g4901/AFMa247wg9/D1S2680/ 646 9-1602-200 1q43 g1262/AFM088xe5/D1S2850 g17846/RH12368/stSG3536 g18893/RH34172/L18266/SHGC-4752 g307/WI-11654/RH50099/RH63795/R10130 g317/EST382595/RH49984/SGC34592 g402/D1S1680/G09467 g421/WI-12850/RH50542/Z41492 g4882/AFMa245wd5/D1S2678 g4901/AFMa247wg9/D1S2680/ 647 9-16022-325 5q31.3-q32 g1166/AFM066xf11/D5S396 g20471/RH60098/WI-10312/ 648 9-16023-160 5q31.3-q32 g1166/AFM066xf11/D5S396 g20471/RH60098/WI-10312/ 649 9-16030-317 5q31.3-q32 g1166/AFM066xf11/D5S396 g20471/RH60098/WI-10312/ 650 9-1605-112 1q43 g1262/AFM088xe5/D1S2850 g17846/RH12368/stSG3536 g18893/RH34172/L18266/SHGC-4752 g307/WI-11654/RH50099/RH63795/R10130 g317/EST382595/RH49984/SGC34592 g402/D1S1680/G09467 g421/WI-12850/RH50542/Z41492 g4882/AFMa245wd5/D1S2678 g4901/AFMa247wg9/D1S2680/ 651 9-1607-373 1q43 g1262/AFM088xe5/D1S2850 g17846/RH12368/stSG3536 g18893/RH34172/L18266/SHGC-4752 g307/WI-11654/RH50099/RH63795/R10130 g317/EST382595/RH49984/SGC34592 g402/D1S1680/G09467 g421/WI-12850/RH50542/Z41492 g4882/AFMa245wd5/D1S2678 g4901/AFMa247wg9/D1S2680/ 652 9-1611-382 1q43 g313/D1S3401/G04332 g404/D1S3450/G11836/ 658 9-1615-118 1q43 g313/D1S3401/G04332 g404/D1S3450/G11836/ 667 9-1622-158 1q43 g2259/AFM218zb6/D1S321/ 670 9-1623-145 1q43 g2259/AFM218zb6/D1S321/ 677 9-16308-315 15q23 g27865/D15S1242/ 680 9-1637-345 1q43 g2259/AFM218zb6/D1S321/ 682 9-1638-571 1q43 g2259/AFM218zb6/D1S321/ 748 9-1701-39 8p23.2-p23.1 g480/WI-9756/G05400/ 758 9-1709-597 8p23.2-p23.1 g480/WI-9756/G05400/ 761 9-1710-249 8p23.2-p23.1 g480/WI-9756/G05400/ 908 9-18130-258 20p12 g1846/AFM197xb12/D20S112/ 931 9-18303-79 2q35 g16358/WI-15771/EST226018/WI-15771/RH56329/ R54614 g879/EST387886/RH56672/SGC32531 g895/WI-15771/EST226018/R54614 g897/WI-19704 g900/WI-20003/RH56649/T19369/ 936 9-18341-95 2q35 g11465/WI-11020/EST206594/WI-11020/RH56985/ R36533 g24950/SHGC-6253/G02482 g891/EST165729/SGC33785/T95608 g892/RH56759/NIB1635/T16652 g893/WI-11020/EST206594/R36533 g894/WI-14333/EST228327/RH57030/R44333 g896/WI-22153/RH56193/ 937 9-18344-284 2q35 g11465/WI-11020/EST206594/WI-11020/RH56985/ R36533 g24950/SHGC-6253/G02482 g891/EST165729/SGC33785/T95608 g892/RH56759/NIB1635/T16652 g893/WI-11020/EST206594/R36533 g894/WI-14333/EST228327/RH57030/R44333 g896/WI-22153/RH56193/ 938 9-18345-107 2q35 g11465/WI-11020/EST206594/WI-11020/RH56985/ R36533 g24950/SHGC-6253/G02482 g891/EST165729/SGC33785/T95608 g892/RH56759/NIB1635/T16652 g893/WI-11020/EST206594/R36533 g894/WI-14333/EST228327/RH57030/R44333 g896/WI-22153/RH56193/ 939 9-18371-433 2q33 g678/WI-14342/R43945/ 940 9-18373-27 2q33 g678/WI-14342/R43945/ 941 9-18375-237 2q33 g678/WI-14342/R43945/ 942 9-18379-485 2q33 g678/WI-14342/R43945/ 973 9-18602-241 1q42.3-q43 g312/D1S3398/G04103/ 974 9-18606-324 1q42.3-q43 g312/D1S3398/G04103/ 976 9-18612-184 1q42.3-q43 g312/D1S3398/G04103/ 977 9-18618-455 2q34 g5200/AFMa351zd1/D2S2242/ 978 9-18620-125 2q34 g5200/AFMa351zd1/D2S2242/ 982 9-18648-71 2q34-q35 g2044/AFM205yb4/D2S295/ 986 9-18715-172 17q23-q24 g1684/AFM168xd12/D17S794/ 987 9-18719-225 2q34-q35 g6159/AFMc009yh1/D2S2322/ 988 9-18720-235 2q34-q35 g6159/AFMc009yh1/D2S2322/ 989 9-18721-442 2q34-q35 g6159/AFMc009yh1/D2S2322/ 993 9-18744-170 2p13-p12;2q35 g731/D2S2722/ 994 9-18745-423 2p13-p12;2q35 g731/D2S2722/ 995 9-18747-72 2p13-p12;2q35 g731/D2S2722/ 1002 9-18808-155 11q23-q24 g4760/AFMa222xc5/D11S4104/ 1003 9-18814-275 18q12.3-q21.1 g1510/AFM147yf2/D18S1094/ 1017 9-18974-99 8p23.1 g21326/RH62553/T03554/IB46 g23/SHGC-9737/G13478 g26491/SHGC-33472/R01769/ 1018 9-18976-135 8p23.1 g21326/RH62553/T03554/IB46 g23/SHGC-9737/G13478 g26491/SHGC-33472/R01769/ 1019 9-18982-345 8p23.1 g21326/RH62553/T03554/IB46 g23/SHGC-9737/G13478 g26491/SHGC-33472/R01769/ 1020 9-18986-248 8p23.1 g21326/RH62553/T03554/IB46 g23/SHGC-9737/G13478 g26491/SHGC-33472/R01769/ 1021 9-18987-191 4q12-q13.1 g3031/AFM292xe1/D4S1592 g3712/AFMa044tf1/D4S3019/ 1022 9-18995-300 4q12-q13.1 g3031/AFM292xe1/D4S1592 g3712/AFMa044tf1/D4S3019/ 1023 9-18996-388 4q12-q13.1 g3031/AFM292xe1/D4S1592 g3712/AFMa044tf1/D4S3019/ 1031 9-19253-102 4q12 g11759/WI-11762/EST183347/RH59610/R12768 g14673/D452603/MR10551 g20013/RH59475/SGC35370/ 1032 9-19256-149 4q12 g11759/WI-11762/EST183347/RH59610/R12768 g14673/D4S2603/MR10551 g20013/RH59475/SGC35370/ 1041 9-1964-53 1q43 g15623/D1S547/GATA4A09 g17350/RH1290/SHGC-477 g17708/RH11033/D29436/D29436 g18500/RH26479/R65593 g319/EST161941/RH50228/RH64454/SGC33718/ T91820 g405/D1S1707/G02394 g408/AFM214xe11/Z66804 g417/D1S2421/WI-9317/D29955/RH49709 g420/WI-15754/RH49799 g426/WI-13731/RH49867/RH64343/R44970/ 1042 9-1977-440 1q43 g15623/D1S547/GATA4A09 g17350/RH1290/SHGC-477 g17708/RH11033/D29436/D29436 g18500/RH26479/R65593 g319/EST161941/RH50228/RH64454/SGC33718/ T91820 g405/D1S1707/G02394 g408/AFM214xe11/Z66804 g417/D1S2421/WI-9317/D29955/RH49709 g420/WI-15754/RH49799 g426/WI-13731/RH49867/RH64343/R44970/ 1044 9-19999-92 4p14;8p22 g1951/AFM200yc7/D4S1547/ 1046 9-20000-252 4p14;8p22 g1951/AFM200yc7/D4S1547/ 1076 9-20294-274 4p14 g5753/AFMb319ze5/D4S2974/ 1077 9-20303-127 4p14 g5753/AFMb319ze5/D4S2974/ 1078 9-20313-311 4p14 g5753/AFMb319ze5/D4S2974/ 1079 9-20320-321 4p14 g19996/RH59112/R77106/SGC34270/ 1080 9-20326-130 4p14 g19996/RH59112/R77106/SGC34270/ 1081 9-20332-432 4p14 g19996/RH59112/R77106/SGC34270/ 1082 9-20335-48 4p14 g19996/RH59112/R77106/SGC34270/ 1083 9-20340-161 4p14 g19996/RH59112/R77106/SGC34270/ 1090 9-20385-215 4p14;8p22 g1951/AFM200yc7/D4S1547/ 1099 9-20469-213 4p14 g12593/WI-14683/EST283388/RH59148 g25359/SHGC4-1576/Z23989/ 1101 9-20480-233 4p14 g12593/WI-14683/EST283388/RH59148 g25359/SHGC4-1576/Z23989/ 1102 9-20481-131 4p14 g12593/WI-14683/EST283388/RH59148 g25359/SHGC4-1576/Z23989/ 1103 9-20485-269 4p14 g12593/WI-14683/EST283388/RH59148 g25359/SHGC4-1576/Z23989/ 1104 9-20493-238 4p14 g12593/WI-14683/EST283388/RH59148 g25359/SRGC4-1576/Z23989/ 1105 9-20499-364 4p14 g12593/WI-14683/EST283388/RH59148 g25359/SHGC4-1576/Z23989/ 1106 9-20504-90 4p14 g12593/WI-14683/EST283388/RH59148 g25359/SHGC4-1576/Z23989/ 1107 9-20508-456 4p14 g12593/WI-14683/EST283388/RH59148 g25359/SRGC4-1576/Z23989/ 1109 9-20511-221 4p14 g12593/WI-14683/EST283388/RH59148 g25359/SHGC4-1576/Z23989/ 1110 9-20514-71 4p14 g12593/WI-14683/EST283388/RH59148 g25359/SHGC4-1576/Z23989/ 1180 9-20938-256 6q22.1-q22.2 g2096/AFM207xb6/D6S412/ 1182 9-20950-251 2p21-p16 g19180/RH56202/L47574/WI-18791/ 1184 9-21012-277 6q12-q13 g6016/AFMb352wc1/D6S1659/ 1185 9-21021-273 6q12-q13 g6016/AFMb352wc1/D6S1659/ 1198 9-2117-107 21q22.1 g1505/AFM147xb12/D21S260 g7678/D21S12 g7852/D21S1824 g7867/D21S1839/ 1199 9-21221-96 3p24.3-p25.1 g24997/D3S4113/ 1221 9-2209-111 10p12.1-p11.2 g1397/AFM119xh12/D10S197/ 1228 9-2214-148 10p12.1-p11.2 g1397/AFM119xh12/D10S197/ 1233 9-2218-219 10p12.1-p11.2 g1397/AFM119xh12/D10S197/ 1235 9-2219-245 10p12.1-p11.2 g1397/AFM119xb12/D10S197/ 1239 9-2220-300 10p12.1-p11.2 g1397/AFM119xh12/D10S197/ 1240 9-22209-304 11q22.3-q23.1 g22439/RH51746/WI-14282/ 1243 9-2222-459 10p12.1-p11.2 g1397/AFM119xh12/D10S197/ 1245 9-22255-384 20q13.1-q13.2 g17162/WI-31010/EST384826/RH57250/RH64058/ SGC31010/ 1246 9-22262-331 19q13.1 g11280/TIGR-A005D28/RH55812 g11653/WI-11537/EST180053/RH56022/R09757/ 1249 9-2228-301 21q21 g7672/D21S11/ 1250 9-2229-240 21q21 g7672/D21S11/ 1254 9-2235-499 21q21 g7672/D21S11/ 1257 9-2240-281 21q22.1 g7689/D21S1230/ 1259 9-2242-206 21q22.1 g7689/D21S1230/ 1260 9-2244-83 21q22.1 g7689/D21S1230/ 1261 9-22442-147 1p31.3-p31.2 g18161/RH17161 g18429/RH25834/Z45206/ 1262 9-22449-216 1p31.3-p31.2 g18161/RH17161 g18429/RH25834/Z45206/ 1263 9-22453-370 1p31.3-p31.2 g18161/RH17161 g18429/RH25834/Z45206/ 1264 9-22456-55 1p31.3-p31.2 g18161/RH17161 g18429/RH25834/Z45206/ 1265 9-2246-340 21q22.1 g7689/D21S1230/ 1266 9-2248-76 21q22.1 g7689/D21S1230/ 1269 9-2250-236 21q22.1 g7689/D21S1230/ 1274 9-22546-125 3q26.2-q26.3 g16578 g19824/RH58812/R39509/WI-11077/ 1275 9-22565-114 14q24.3-q31 g23143/RH53688/G04281/WI-3377 g27731/D14S929/ 1276 9-22571-136 14q24.3-q31 g23143/RH53688/G04281/WI-3377 g27731/D14S929/ 1282 9-22604-208 1p32.1-p31.3 g11032/RH49618/NIB551/T17225 g12546/WI-14549/EST42945/RH50683/T17225/ 1283 9-22610-343 15q21 g12995/WI-15792/EST265261/RH54066 g27831/SHGC-9535/ 1284 9-22615-392 15q21 g12995/WI-15792/EST265261/RH54066 g27831/SHGC-9535/ 1285 9-22617-378 15q21 g12995/WI-15792/EST265261/RH54066 g27831/SHGC-9535/ 1286 9-22620-404 19q13.4 g12950/WI-15666/EST230907/RH55986/R44502/ 1287 9-22628-292 15q24 g16003/WI-20012/EST48352/RH54344/T28573 g23432/RH54202/WI-7231/ 1288 9-22629-124 15q24 g16003/WI-20012/EST48352/RH54344/T28573 g23432/RH54202/WI-7231 1289 9-22632-237 15q24 g16003/WI-20012/EST48352/RH54344/T28573 g23432/RH54202/WI-7231/ 1290 9-22646-233 6q21 g5686/AFMb312yc1/D6S1635/ 1291 9-22648-57 6q21 g5686/AFMb312yc1/D6S1635/ 1292 9-22650-64 6q21 g5686/AFMb312yc1/D6S1635/ 1293 9-22652-343 8q24.2 g5926/AFMb340xd5/D8S1783/ 1294 9-22655-319 8q24.2 g5926/AFMb340xd5/D8S1783/ 1295 9-22660-386 8q24.2 g5926/AFMb340xd5/D8S1783/ 1296 9-22662-268 8q24.2 g5926/AFMb340xd5/D8S1783/ 1297 9-22666-164 4p15.3-p15.2 g19966/RH58930/L14153/D4S1267/ 1298 9-22668-232 4p15.3-p15.2 g19966/RH58930/L14153/D4S1267/ 1299 9-22674-31 4p15.3-p15.2 g19966/RH58930/L14153/D4S1267/ 1300 9-22675-187 4p15.3-p15.2 g19966/RH58930/L14153/D4S1267/ 1301 9-22680-130 18p11.31 g23841/RH55516/G03618/WI-4219/ 1302 9-22683-107 18p11.31 g23841/RH55516/G03618/WI-4219/ 1304 9-22700-358 11q23.1-q23.2; g27251/SHGC-2090/Z24364/RH13666 5q11.2 g3346/AFM320Xh1/D11S1347/ 1305 9-22701-307 11q23.1-q23.2; g27251/SHGC12090/Z24364/RH13666 5q11.2 g3346/AFM320xh1/D11S1347/ 1312 9-22733-281 2q35 g19425/RH56275/H62242/SGC32398/RH56275/ 1313 9-22741-180 2q34-q35 g6159/AFMc009yh1/D2S2322/ 1316 9-22771-150 2q35 g24953/SRGC-971/Z17049 g4193/AFMa109wg5/D2S2151/ 1317 9-22775-365 2q35 g24953/SHGC-971/Z17049 g4193/AFMa109wg5/D2S2151/ 1319 9-22785-431 2q35 g24953/SHGC-971/Z17049 g4193/AFMa109wg5/D2S2151/ 1320 9-22843-342 2q35 g874/WI-12463/EST195179/R25134/ 1321 9-22844-211 2q35 g874/WI-12463/EST195179/R25134/ 1323 9-22868-425 2q33 g1453/AFM135xf12/D2S2396/ 1324 9-22872-431 2q33 g1453/AFM135xf12/D2S2396/ 1325 9-2288-144 21q21.3 g5554/AFMb291yb9/D21S1896 g7754/D21S1435/ 1326 9-22917-145 2q32.3-q33 g6130/AFMc005wb9/D2S2318/ 1328 9-22948-262 2p23 g10323/AFMB353WF1/w2082/ 1329 9-22954-306 2p23 g10323/AFMB353WF1/w2082/ 1330 9-22957-409 2p23 g10323/AFMB353WF1/w2082/ 1331 9-22959-239 2p23 g10323/AFMB353WF1/w2082/ 1332 9-22964-82 11p15.5-p15.4 g6052/AFMb355za9/D11S4177/ 1333 9-22975-126 11p15.5-p15.4 g6052/AFMb355za9/D11S4177/ 1334 9-23014-300 2q34-q35 g2044/AFM205yb4/D2S295/ 1335 9-23018-166 2q34-q35 g2044/AFM205yb4/D2S295/ 1336 9-23020-187 1q43 g422/WI-15487/RH50392/RH64322/R39926 g431/WI-31075/RH50186/RH64283/SGC31075/ 1337 9-23083-59 1p34.2-p34.1 g18865/RH33933/G07594/SHGC-4031/ 1338 9-23100-367 9q33-q34.1 g15044/D9S1698/HSC0VC072/ 1339 9-23115-404 22q12;2p23 g19151/RH57021/R95095/SGC33508 g19152/RH56155/WI-10842/ 1340 9-23118-402 22q12;2p23 g19151/RH57021/R95095/SGC33508 g19152/RH56155/WI-10842/ 1341 9-2312-358 21q21.2 g7697/D21S1240/ 1342 9-23123-250 3p21.1-p14.3 g2744/AFM268wg9/D3S1578/ 1343 9-23127-314 3p21.1-p14.3 g2744/AFM268wg9/D3S1578/ 1344 9-23132-192 3p21.1-p14.3 g2744/AFM268wg9/D3S1578/ 1345 9-23134-89 3p21.1-p14.3 g2744/AFM268wg9/D3S1578/ 1346 9-2315-213 21q21.2 g7697/D21S1240/ 1347 9-23150-262 18q23 g13666/WI-18089/EST355463/RH55492 g28291/SHGC-17251/ 1348 9-2320-292 21q21.2 g7697/D21S1240/ 1349 9-23201-345 4q31.1-q31.2 g16662/WI-15195/EST308096/RH59447 g25614/D4S507 g6094/AFMb361zg5/D4S2998/ 1350 9-23202-185 4q31.1-q31.2 g16662/WI-15195/EST308096/RH59447 g25614/D4S507 g6094/AFMb361zg5/D4S2998/ 1351 9-23204-262 4q31.1-q31.2 g16662/WI-15195/EST308096/RH59447 g25614/D4S507 g6094/AFMb361zg5/D4S2998/ 1352 9-23207-281 4q31.1-q31.2 g16662/WI-15195/EST308096/RH59447 g25614/D4S507 g6094/AFMb361zg5/D4S2998/ 1353 9-2321-82 21q21.2 g7697/D21S1240/ 1354 9-23228-176 2q21 g19179/RH56626/T10467/SGC32727 g19285/RH56961/R51826/SGC31844/ 1355 9-2324-338 21q21.2 g7697/D21S1240/ 1358 9-2328-535 10p11.2 g1912/AFM199zb6/D10S213/ 1359 9-23299-424 3q21 g5752/AFMb319yf1/D3S3646/ 1360 9-23302-326 3q21 g5752/AFMb319yf1/D3S3646/ 1361 9-2331-639 21q22.1 g7774/D21S1677 g7876/D21S1853 g7877/D21S1854 g7886/D21S1865 g7887/D21S1866/ 1362 9-23312-93 3q21 g5752/AFMb319yf1/D3S3646/ 1363 9-23317-51 1q32.1 g5230/AFMb002ya5/D1S2716/ 1364 9-23322-49 1q32.1 g5230/AFMb002ya5/D1S2716/ 1365 9-23326-120 1q32.1 g5230/AFMb002ya5/D1S2716/ 1366 9-23328-292 1q32.1 g5230/AFMb002ya5/D1S2716/ 1367 9-23333-157 9p22 g10403/D95921/GATA-D9S921 g26676/SHGC-3751/Z16707/RH13288/ 1368 9-23334-443 9p22 g10403/D9S921/GATA-D9S921 g26676/SHGC-3751/Z16707/RH13288/ 1369 9-23359-99 2q35 g878/RH56992/SGC35345/ 1370 9-23381-412 2q34 g5200/AFMa351zd1/D2S2242/ 1371 9-23387-404 2q34 g5200/AFMa351zd1/D2S2242 / 1372 9-23413-242 11q23.3-q24 g2707/AFM265wa9/D11S1328/ 1373 9-23415-131 11q23.3-q24 g2707/AFM265wa9/D11S1328/ 1374 9-23417-128 11q23.3-q24 g2707/AFM265wa9/D11S1328/ 1378 9-2345-28 13p13-q11; g6058/AFMb356wg1/D21S1904 14p13-q11.1; g7727/D21S13 15p13-q11.1; g7737/D21S1416 16p11.1-q11.2; g7750/D21S1431/ 1p11-q12; 21p13-q11.1; 3p11-q11.1; 9p11-q12; Yq12 1383 9-23462-192 1q21.1-q21.2 g17255/RH421/Z24671/SHGC-1599/RH13725 g19054/RH36179/H47260/stSG12720/ 1384 9-23463-118 1q21.1-q21.2 g17255/RH421/Z24671/SHGC-1599/RH13725 g19054/RH36179/H47260/stSG12720/ 1386 9-2347-207 13p13-q11; g6058/AFMb356wg1/D21S1904 14p13-q11.1; g7727/D21S13 15p13-q11.1; g7737/D21S1416 16p11.1-q11.2; g7750/D21S1431/ 1p11-q12; 21p13-q11.1; 3p11-q11.1; 9p11-q12; Yq12 1388 9-2348-127 13p13-q11; g6058/AFMb356wg1/D21S1904 14p13-q11.1; g7727/D21S13 15p13-q11.1; g7737/D21S1416 16p11.1-q11.2; g7750/D21S1431/ 1p11-q12; 21p13-q11.1; 3p11-q11.1; 9p11-q12; Yq12 1394 9-2356-322 13p13- g6058/AFMb3S6wg1/D21S1904 q11;14p13- g7727/D21S13 q11.1;15p13- g7737/D21S1416 q11.1;16p11.1- g7750/D21S1431/ q11.2;1p11- q12;21p13- q11.1;3p11- q11.1;9p11- q12;Yq12 1395 9-2362-270 21q21-q22.1 g7899/D21S1877 g7904/D21S1881/ 1396 9-2364-329 21q21-q22.1 g7899/D21S1877 g7904/D21S1881/ 1397 9-2367-61 21q21-q22.1 g7899/D21S1877 g7904/D21S1881/ 1398 9-2368-61 21q21-q22.1 g7899/D21S1877 g7904/D21S1881/ 1399 9-23687-107 2q34 g2193/AFM212ze9/D2S157/ 1400 9-237-151 21q22.3 g7967/D21S49/ 1401 9-23714-196 15q23 g27865/D15S1242/ 1403 9-2375-114 21q11.2 g7010/D21S258 g7029/AFMa083xe1/ 1405 9-2378-200 21q11.2 g7010/D21S258 g7029/AFMa083xe1/ 1406 9-2381-394 21q11.2 g7010/D21S258 g7029/AFMa083xe1/ 1409 9-2413-368 21q22.1 g7798/D21S1700 g7879/D21S1856/ 1410 9-2417-177 21q22.1 g7798/D21S1700 g7879/D21S1856/ 1411 9-2419-285 21q22.1 g7798/D21S1700 g7879/D21S1856/ 1412 9-24246-247 2q33 g649/Mch4 g651/RH15884/T91183/ 1413 9-24253-437 2q35 g15152/D2S2592/UTR-05171/ 1414 9-24259-466 2q35 g15152/D2S2592/UTR-05171/ 1415 9-24264-380 2q35 g15152/D2S2592/UTR-05171/ 1416 9-24269-417 2q33-q34 g2141/AFM210yf10/D2S155/ 1417 9-24270-207 2q33-q34 g2141/AFM210yf10/D2S155/ 1419 9-24284-213 2q33-q34 g732/RH56861/ 1420 9-24286-231 2q33-q34 g732/RH56861/ 1421 9-24288-121 2q33-q34 g732/RH56861/ 1422 9-24333-37 2q34 g882/EST141512/RH56286/SGC33506/T79149/ 1423 9-24342-311 2q35 g679/EST250412/RH56354/SGC31996/ 1424 9-24376-24 7q11.23-q21.1 g11961/WI-12513/EST251317/RH61608/R74459/ 1425 9-24379-319 7q11.23-q21.1 g11961/WI-12513/EST251317/RH61608/R74459/ 1429 9-24390-27 10q11.2 g21928/RH51060/SGC38063/ 1430 9-24392-61 10q11.2 g21928/RH51060/SGC38063/ 1431 9-24393-108 10q11.2 g21928/RH51060/SGC38063/ 1433 9-24409-383 14q24.3 g23123/RH53961/T40920/SGC32981/SHGC-32981/ 1434 9-24411-420 14q24.3 g23123/RH53961/T40920/SGC32981/SHGC-32981/ G27696/G25597/EST91724 g27725/SHGC-942/Z16981/RH49039/ 1435 9-24427-321 5q34 g1927/AFM200vf6/D5S619/ 1436 9-24432-284 5q34 g1927/AFM200vf6/D5S619/ 1439 9-24447-448 1p31.3-p31.2 g14348/D1S2161/MR7398 g18605/RH27953/G07842/ 1441 9-24454-257 1p31.3-p31.2 g14348/D1S2161/MR7398 g18605/RH27953/G07842/ 1442 9-24463-206 21q21-q22.1 g24227/RH57563/H92581/WI-22816 g7742/D21S1422 1443 9-24496-171 11q23.3-q24 g22500/RH52371/H30529/SGC30738 1444 9-24506-396 4q25 g20076/RH59480/Z15005/WI-6987 g20078/RH59042/T16396/WI-13405/ 1445 9-24508-45 4q25 g20076/RH59480/Z15005/WI-6987 g20078/RH59042/T16396/WI-13405/ 1446 9-24529-330 2q35 g16358/WI-15771/EST226018/WI-15771/RH56329/ R54614 g879/EST387886/RH56672/SGC32531 g895/WI-15771/EST226018/R54614 g897/WI-19704 g900/WI-20003/RH56649/T19369/ 1447 9-24534-317 2q35 g16358/WI-15771/EST226018/WI-15771/RH56329/ R54614 g879/EST387886/RH56672/SGC32531 g895/WI-15771/EST226018/R54614 g897/WI-19704 g900/WI-20003/RH56649/T19369/ 1448 9-24554-324 2q35 g875/WI-12994/HSC2KC082 g876/WI-17411/EST240716/RH56866/R63961/ 1449 9-24557-406 2q35 g5212/AFMa357wc9/D2S2244/ 1450 9-24561-360 2q35 g13275/WI-16791/EST159864/WI-16791/RH56414/ T89743 g889/WI-16791/EST159864/T89743/ 1451 9-24570-260 6q26 g15128/D6S1951/UTR-00083/ 1453 9-24725-138 16q21-q22; g12359/WI-13905/EST227346/RH54662/R49366 7p11.1-q11.1 g16052/WI-20039/EST59759/RH54768/T33895 g27992/SHGC-11618/T56923/ 1454 9-24727-360 16q21-q22; g12359/WI-13905/EST227346/RH54662/R49366 7p11.1-q11.1 g16052/WI-20039/EST59759/RH54768/T33895 g27992/SHGC-11618/T56923/ 1455 9-24750-293 11q23.1-q23.2 g15768 g22459/RH52046/H86791/SGC31226/ 1456 9-24778-221 7q22 g491/WI-6368/RH61376/ 1457 9-24793-390 7q22 g491/WI-6368/RH61376/ 1458 9-24800-565 7q22 g491/WI-6368/RH61376/ 1461 9-25053-114 2q33-q34 g6158/AFMc009wh1/D2S2321/ 1462 9-25055-44 2q33-q34 g6158/AFMc009wh1/D2S2321/ 1463 9-25070-78 2q33-q34 g6158/AFMc009wh1/D2S2321/ 1467 9-2524-98 21q22.3 g7967/D21S49/ 1468 9-25246-170 13q34 g15911 g712/RH17028/ 1469 9-25249-151 13q34 g15911 g712/RH17028/ 1470 9-2525-142 21q22.3 g7967/D21S49/ 1471 9-25255-288 13q34 g15911 g712/RH17028/ 1472 9-25369-121 15q21 g23347/RH54301/G05439/WI-9836 g23349/RH54281/H92576/SGC32630/ 1478 9-25431-269 2q33-q34 g881/WI-18179/EST362695/ 1479 9-25432-119 2q33-q34 g881/WI-18179/EST362695/ 1480 9-25433-351 2q33-q34 g881/WI-18179/EST362695/ 1481 9-25447-272 1q44 g17338/RH1233/SHGC-269 g18240/RH17889/R98962/R98962 g18593/RH27933/RH64496/ 1482 9-25448-348 18q11.2 g10695/CHLC.GATA84D10/CHLC.GATA85D10.P19280/ G08009/GATA-P19280/ 1483 9-25452-83 18q11.2 g10695/CHLC.GATA85D10/CHLC.GATA85D10.P19280/ G08009/GATA-P19280/ 1484 9-25454-349 18q11.2 g10695/CHLC.GATA85D10/CHLC.GATA85D10.P19280/ G08009/GATA-P19280/ 1500 9-2570-218 21q22.3 g24258/RH57551/R48588/SGC34143 g24259/RH57619/H53556/SGC34732/ 1502 9-25716-393 13q34 g866/WI-13756/RH53429/R46080/ 1503 9-25717-252 13q34 g866/WI-13756/RH53429/R46080/ 1507 9-25781-275 19q13.2 g11925/WI-12417/EST276107/RH55689/ 1509 9-2597-34 21q22.2 g7732/D21S1411/ 1510 9-26001-224 3q27-q28 g11074/RH58557/Z22625/T40957 g19863/RH58270/H61445/SGC34843/ 1511 9-26002-93 3q27-q28 g11074/RH58557/Z22625/T40957 g19863/RH58270/H61445/SGC34843/ 1512 9-26042-310 2q23 g1807/AFM191wg9/D2S142/ 1513 9-26080-152 11q24;5q15 g4300/AFMa124wg5/D5S1957/ 1514 9-26082-48 11q24;5q15 g4300/AFMa124wg5/D5S1957/ 1515 9-26099-119 1p22 g17899/RH12743/R47991/stSG4580/ 1517 9-26105-273 2q35 g19425/RH56275/H62242/SGC32398/RH56275/ 1518 9-26116-191 5q21-q22 g13747/WI-18306/EST374223/RH59791 g20406/RH60017/H10223/SGC32412/ 1521 9-2624-407 21q22.2 g16433 g24246/RH57580/SGC32448 g28577/SHGC-10474 g28580/SHGC-10477 g7818/D21S1725E/ 1522 9-2625-70 21q22.2 g16433 g24246/RH57580/SGC32448 g28577/SHGC-10474 g28580/SHGC-10477 g7818/D21S1725E/ 1523 9-2637-28 21q22.2 g16433 g24246/RH57580/SGC32448 g28577/SHGC-10474 g28580/SHGC-10477 g7818/D21S1725E/ 1528 9-342-288 19q13.2-q13.3 g448/RH11470/RH1669/ 1544 9-449-344 10p12.1-p11.2 g3518/AFM338ta5/D10S600/ 1545 9-4536-255 15q14-q15 g15965/WI-11934/EST197813/RH54176/R27768 g15966/WI-19599/ 1546 9-4541-39 1p36.2-p36.1 g316/EST47321/D19656/RH50110/SGC32758 g414/WI-12386/RH50879/RH63782/ 1547 9-4544-287 1p36.2-p36.1 g316/EST47321/D19656/RH50110/SGC32758 g414/WI-12386/RH50879/RH63782/ 1548 9-4547-312 1p36.2-p36.1 g316/EST47321/D19656/RH50110/SGC32758 g414/WI-12386/RH50879/RH63782/ 1549 9-4595-341 1q43 g314/WI-10464/ 1550 9-4604-26 1q43 g314/WI-10464/ 1555 9-465-443 10p12.1-p11.2 g1621/AFM164tg9/D10S204/ 1557 9-466-361 10p12.1-p11.2 g1621/AFM164tg9/D10S204/ 1569 9-472-70 10p11.2 g2598/AFM254xb1/D10S224/ 1591 9-490-202 10p12.1-p11.2 g5171/AFMa346zd5/D10S1695 g5966/AFMb345ya9/D10S1732/ 1594 9-4950-196 1q43 g416/D1S3481/G13394/ 1595 9-4951-36 1q43 g416/D1S3481/G13394/ 1599 9-5016-206 7q11.23-q21.1 g1970/AFM203vb6/D7S634/ 1600 9-5029-240 7q11.23-q21.1 g1970/AFM203vb6/D7S634/ 1605 9-5099-245 7q11.23-q21.1 g2935/AFM286xf9/D7S669/ 1606 9-5101-284 7q11.23-q21.1 g2935/AFM286xf9/D7S669/ 1607 9-5104-160 7q11.23-q21.1 g2935/AFM286xf9/D7S669/ 1608 9-5107-184 7q11.23-q21.1 g2935 /AFM286xf9/D7S669/ 1609 9-5108-144 7q11.23-q21.1 g2935/AFM286xf9/D7S669/ 1610 9-511-33 10p12.1-p11.2 g4171/AFMa106vf5/D10S1639/ 1633 9-5294-362 2q33 g4971/AFMa28Szb9/D2S2214 g4987/AFMa289xc1/D2S2217/ 1634 9-5306-93 2q33 g4971/AFMa285zb9/D2S2214 g4987/AFMa289xc1/D2S2217/ 1635 9-5308-341 2q33 g4971/AFMa285zb9/D2S2214 g4987/AFMa289xc1/D2S2217/ 1636 9-5312-273 2q33 g4971/AFMa285zb9/D2S2214 g4987/AFMa289xc1/D2S2217/ 1639 9-5355-165 1q43 g430/RH50609/RH71064/SGC35584/ 1640 9-5356-100 1q43 g430/RH50609/RH71064/SGC35584/ 1641 9-5360-151 1q43 g430/RH50609/RH71064/SGC35584/ 1642 9-5362-203 1q43 g430/RH50609/RH71064/SGC35584/ 1643 9-5364-95 1q43 g430/RH50609/RH71064/SGC35584/ 1644 9-5379-158 2p13 g681/WI-9025/ 1645 9-5386-85 2p13 g681/WI-9025/ 1654 9-5420-425 1q43 g496/WI-20654/TWIK1/EST/RH50086/T89039/ 1655 9-5427-466 1q43 g496/WI-20654/TWIK1/EST/RH50086/T89039/ 1656 9-5432-391 1q43 g496/WI-20654/TWIK1/EST/RH50086/T89039/ 1657 9-5433-45 1q43 g496/WI-20654/TWIK1/EST/RH50086/T89039/ 1658 9-5437-159 1q43 g496/WI-20654/TWIK1/EST/RH50086/T89039/ 1659 9-5438-70 1q43 g496/WI-20654/TWIK1/EST/RH50086/T89039/ 1660 9-5441-287 1q43 g496/WI-20654/TWIK1/EST/RH50086/T89039/ 1661 9-5446-303 1q43 g496/WI-20654/TWIK1/EST/RH50086/T89039/ 1662 9-5447-322 1q43 g496/WI-20654/TWIK1/EST/RH50086/T89039/ 1663 9-5458-203 2q32.3 g2869/AFM280wd5/D2S342/ 1664 9-5468-319 2q33.3-q34 g1197/AFM074xg9/D2S307 g24934/SHGC-3548/Z23329/ 1665 9-5472-290 2q34-q35 g4057/AFMa082xc5/D2S2382 g680/D2S2606/XRCC5?/ 1666 9-5475-455 2q34-q35 g4057/AFMa082xc5/D2S2382 g680/D2S2606/XRCC5?/ 1667 9-5477-207 2q34-q35 g4057/AFMa082xc5/D2S2382 g680/D2S2606/XRCC5?/ 1668 9-5485-325 2q34-q35 g4057/AFMa082xc5/D2S2382 g680/D2S2606/XRCC5?/ 1669 9-5490-368 2q34-q35 g4057/AFMa082xc5/D2S2382 g680/D2S2606/XRCC5?/ 1670 9-5494-205 2q34-q35 g4057/AFMa082xc5/D2S2382 g680/D2S2606/XRCC5?/ 1671 9-5502-433 2q34-q35 g4057/AFMa082xc5/D2S2382 g680/D2S2606/XRCC5?/ 1672 9-5505-226 2q33 g19410/RH56788/H90757/SGC35219 g50057/RH67919 g733/WI-8988/D2S2634/G07066/ 1673 9-5516-121 2q33 g19410/RH56788/H90757/SGC35219 g50057/RH67919 g733/WI-8988/D2S2634/G07066/ 1674 9-5526-334 2q33 g19410/RH56788/H90757/SGC35219 g50057/RH67919 g733/WI-8988/D2S2634/G07066/ 1675 9-5566-131 2q33 g675/RH18054/ 1676 9-5582-71 2q34-q35 g19417/RH56312/R44983/SGC31824 g646/RH56681/SGC33209/R02572 g647/D2S2635 g887/WI-10220/EST135001/T72644/ 1677 9-5590-99 2q34-q35 g19417/RH56312/R44983/SGC31824 g646/RH56681/SGC33209/R02572 g647/D2S2635 g887/WI-10220/EST135001/T72644/ 1678 9-5595-380 2q34-q35 g19417/RH56312/R44983/SGC31824 g646/RH56681/SGC33209/R02572 g647/D2S2635 g887/WI-10220/EST135001/T72644/ 1679 9-5596-216 2q34-q35 g19417/RH56312/R44983/SGC31824 g646/RH56681/SGC33209/R02572 g647/D2S2635 g887/WI-10220/EST135001/T72644/ 1680 9-5604-376 2q34-q35 g19417/RH56312/R44983/SGC31824 g646/RH56681/SGC33209/R02572 g647/D2S2635 g887/WI-10220/EST135001/T72644/ 1681 9-5608-324 2q34-q35 g19417/RH56312/R44983/SGC31824 g646/RH56681/SGC33209/R02572 g647/D2S2635 g887/WI-10220/EST135001/T72644/ 1753 9-6131-166 2q33 g4971/AFMa285zb9/D2S2214 g4987/AFMa289xc1/D2S2217/ 1754 9-6135-319 2q33 g4971/AFMa285zb9/D2S2214 g4987/AFMa289xc1/D2S2217/ 1756 9-6141-339 2q33 g4971/AFMa285zb9/D2S2214 g4987/AFMa289xc1/D2S2217/ 1759 9-6176-96 2q33.3-q34 g1197/AFM074xg9/D2S307 g24934/SHGC-3548/Z23329/ 1760 9-6180-389 2q33.3-q34 g1197/AFM074xg9/D2S307 g24934/SHGC-3548/Z23329/ 1761 9-6181-328 2q33.3-q34 g1197/AFM074xg9/D2S307 g24934/SHGC-3548/Z23329/ 1762 9-6189-224 2p13 g681/WI-9025/ 1764 9-6191-252 2p13 g681/WI-9025/ 1765 9-6193-88 2p13 g681/WI-9025/ 1767 9-6217-420 2q32.3 g2869/AFM280wd5/D2S342/ 1769 9-6253-308 1q42.3-q43 g498/WI-21544/EST/RH50234/R54815/ 1770 9-6257-226 1q42.3-q43 g498/WI-21544/EST/RH50234/R54815/ 1771 9-6261-172 1q42.3-q43 g498/WI-21544/EST/RH50234/R54815/ 1772 9-6278-391 2q35 g2382/AFM234xb8/D2S164 g5267/AFMb009zd5/D2S2248/ 1773 9-6294-184 2q35 g2382/AFM234xb8/D2S164 g5267/AFMb009zd5/D2S2248/ 1774 9-6298-280 2q35 g2382/AFM234xb8/D2S164 g5267/AFMb009zd5/D2S2248/ 1775 9-6300-106 2q35 g2382/AFM234xb8/D2S164 g5267/AFMb009zd5/D2S2248/ 1776 9-6310-217 1q42.3-q43 g498/WI-21544/EST/RH50234/R54815/ 1782 9-6404-147 9q34.2 g2548/AFM248wf1/D9S179 g26813/SHGC-3659/Z17118/ 1783 9-6409-62 9q34.2 g2548/AFM248wf1/D9S179 g26813/SHGC-3659/Z17118/ 1784 9-6411-93 9q34.2 g2548/AFM248wf1/D9S179 g26813/SHGC3659/Z17118/ 1785 9-6413-369 9q34.2 g2548/AFM248wf1/D9S179 g26813/SHGC-3659/Z17118/ 1881 9-7098-382 11q23.3-q24 g2274/AFM220xh6/D11S5924 g5211/AFMa357wa5/D11S4129 g5947/AFMb342ze9/D11S4171/ 1882 9-7103-155 11q23.3-q24 g2274/AFM220xh6/D11S924 g5211/AFMa357wa5/D11S4129 g5947/AFMb342ze9/D11S4171/ 1883 9-7104-187 11q23.3-q24 g2274/AFM220xh6/D11S924 g5211/AFMa357wa5/D11S4129 g5947/AFMb342ze9/D11S4171/ 1884 9-7107-143 11q23.3-q24 g2274/AFM220xh6/D11S924 g5211/AFMa357wa5/D11S4129 g5947/AFMb342ze9/D11S4171/ 1885 9-7114-31 11q23.3-q24 g2274/AFM220xh6/D11S924 g5211/AFMa357wa5/D11S4129 g5947/AFMb342ze9/D11S4171/ 1886 9-719-278 10q25.3-q26.1 g5771/AFMb320zb5/D10S1722/ 1892 9-7141-395 8p21 g12037/WI-12748/RH62389/G13379/HSC2SC022/ 1895 9-7167-438 1q43-q44 g1317/AFM102xe3/D1S204 g5995/AFMb349xb9/D1S2785/ 1896 9-7172-441 1q43-q44 g1317/AFM102xe3/D1S204 g5995/AFMb349xb9/D1S2785/ 1897 9-7177-81 1q43 g12003/WI-12648/EST332992/RH49763/RH63832/ R92197/ 1899 9-7183-338 1q43 g12003/WI-12648/EST332992/RH49763/RH63832/ R92197/ 1900 9-7193-228 1q43 g12003/WI-12648/EST332992/RH49763/RH63832/ R92197/ 1902 9-7212-346 1q43 g12003/WI-12648/EST332992/RH49763/RH63832/ R92197/ 1903 9-7214-109 8p21 g12037/WI-12748/RH62389/G13379/HSC25C022/ 1904 9-7218-444 8p21 g12037/WI-12748/RH62389/G13379/HSC25C022/ 1905 9-7234-101 11q23-q24 g11907/WI-112357/EST142002/RH51673/T79639 g5745/AFMb318z19/D11S4157/ 1907 9-7252-279 11q23.3 g4523/AFMa162wf5/D11S4089/ 1962 9-7671-33 11q24 g5238/AFMb004z19/D11S4132/ 1963 9-7677-107 11q24 g5238/AFMb004z19/D11S4132/ 1964 9-7688-325 11q23.3-q24 g2707/AFM265wa9/D11S1328/ 1965 9-7692-340 11q23.3-q24 g2707/AFM265wa9/D11S1328/ 1967 9-7706-303 11q24 g5443/AFMb066zg9/D11S4144/ 1969 9-7710-318 12q14-q15 g5395/AFMb043wd1/D12S1649/ 1970 9-7712-176 12q14-q15 g5395/AFMb043wd1/D12S1649/ 1971 9-7721-379 12q14-q15 g5395/AFMb043wd1/D12S1649/ 1972 9-7727-65 14q13; g6091/AFMb361yh9/D14S1034/ 15q13-q14 1973 9-7728-334 14q13; g6091/AFMb361yh9/D14S1034/ 15q13-q14 1974 9-7732-122 14q13; g6091/AFMb361yh9/D14S1034/ 15q13-q14 1975 9-7737-264 2q32.1-q32.2 g19365/RH56584/SGC31527 g19370/RH57027/R78360/SGC34224 g2101/AFM207xg1/D2S152/ 1976 9-7744-255 2q32.1-q32.2 g19365/RH56584/SGC31527 g19370/RH57027/R78360/SGC34224 g2101/AFM207xg1/D2S152/ 1977 9-7745-305 2q32.1-q32.2 g19365/RH56584/SGC31527 g19370/RH57027/R78360/SGC34224 g2101/AFM207xg1/D2S152/ 1978 9-7749-123 2q32.1-q32.2 g19365/RH56584/SGC31527 g19370/RH57027/R78360/SGC34224 g2101/AFM207xg1/D2S152/ 1979 9-7751-450 2q33 g5710/AFMb315xd5/D2S2287/ 1980 9-7753-199 2q33 g5710/AFMb315xd5/D2S2287/ 1981 9-7754-119 2q33 g5710/AFMb315xd5/D2S2287/ 1982 9-7759-63 2q33 g5710/AFMb315xd5/D2S2287/ 1983 9-7762-227 2q33 g5710/AFMb315xd5/D2S2287/ 1984 9-7764-161 2q33 g5710/AFMb315xd5/D2S2287/ 1985 9-7775-313 14q32.2 g27781/SHGC-1408/Z23999/ 1986 9-7784-31 11q23.3-q24 g2623/AFM256za5/D11S936 g3466/AFM331yc5/D11S1353/ 1987 9-7789-404 11q23.3-q24 g2623/AFM256za5/D11S936 g3466/AFM331yc5/D11S1353/ 1988 9-7792-173 11q23.3-q24 g2623/AFM256za5/D11S936 g3466/AFM331yc5/D11S1353/ 1989 9-7796-130 2q32.3-q33 g6130/AFMc005wb9/D2S2318/ 1990 9-7803-253 2q32.3-q33 g6130/AFMc005wb9/D2S2318/ 1992 9-7840-281 2q31-q32.1 g5604/AFMb297xc1/D2S2273/ 1994 9-7868-204 2q34-q35 g2794/AFM273va9/D2S334/ 1995 9-7869-135 2q34-q35 g2794/AFM273va9/D2S334/ 1996 9-7870-316 2q34-q35 g2794/AFM273va9/D2S334/ 1997 9-7877-363 2q34-q35 g2794/AFM273va9/D2S334/ 1998 9-7882-43 2q35 g2211/AFM214ye1/D2S301/ 1999 9-7883-411 2q35 g2211/AFM214ye1/D2S301/ 2000 9-7884-151 2q35 g2211/AFM214ye1/D2S301/ 2001 9-7893-226 2q35 g2211/AFM214ye1/D2S301/ 2002 9-7898-43 2q33-q34 g3773/AFMa050ya5/D2S2358/ 2003 9-7900-452 2q33-q34 g3773/AFMa050ya5/D2S2358/ 2005 9-7917-429 2q33-q34 g3773/AFMa050ya5/D2S2358/ 2024 9-806-152 13p13-q11; g7719/D21S1277/ 14p13-q11.1; 15p13-q11.1; 1p11-q12; 21p13; 22p13-q11.1; 9p11-q12 2033 9-810-117 13p13-q11; g7719/D21S1277/ 14p13-q11.1; 15p13-q12; 21p13; 22p13-q11.1; 9p11-q12 2087 9-8453-358 2q32.2 g5488/AFMb082ye1/D2S2262/ 2088 9-8454-152 2q32.2 g5488/AFMb082ye1/D2S2262/ 2089 9-8456-266 2q32.2 g5488/AFMb082ye1/D2S2262/ 2090 9-8457-239 2q32.2 g5488/AFMb082ye1/D2S2262/ 2091 9-8470-275 2q32.2 g5488/AFMb082ye1/D2S2262/ 2092 9-8472-152 2q32.2 g5488/AFMb082ye1/D2S2262/ 2093 9-8476-216 2q34 g2193/AFM212ze9/D2S157/ 2094 9-8478-385 2q34 g2193/AFM212ze9/D2S157/ 2095 9-8487-245 2q34 g2193/AFM212ze9/D2S157/ 2096 9-8491-339 2q34 g2193/AFM212ze9/D2S157/ 2097 9-8499-107 2q34 g2193/AFM212ze9/D2S157/ 2098 9-8505-269 2q36 g643/T95608/SGC33785/EST165729/RH56667 g676/WI-11020/R36533/ 2100 9-8510-44 2q36 g643/T95608/SGC33785/EST165729/RH56667 g676/WI-11020/R36533/ 2101 9-8514-434 2q36 g643/T95608/SGC33785/EST165729/RH56667 g676/WI-11020/R36533/ 2102 9-8530-209 2q36 g643/T95608/SGC33785/EST165729/RH56667 g676/WI-11020/R36533/ 2110 9-8583-146 14q13 g3541/AFM340zd9/D14S1049/ 2111 9-8588-369 14q13 g3541/AFM340zd9/D14S1049/ 2112 9-8590-287 14q13 g3541/AFM340zd9/D14S1049/ 2196 9-921-285 13q31.1 g14575/D13S1196/MR8039 g8824/D13S1196/WI5275/ 2197 9-924-93 13q31.1 g14575/D13S1196/MR8039 g8824/D13S1196/WI5275/ 2200 9-9254-404 2q33.3 g1909/AFM199yf2/D2S2237/ 2201 9-926-98 13q31.1 g14575/D13S1196/MR8039 g8824/D13S1196/WI5275/ 2202 9-9263-283 2q33.3 g1909/AFM199yf2/D2S2237/ 2203 9-9271-70 2q33.3 g1909/AFM199yf2/D2S2237/ 2204 9-9274-246 2q33.3 g1909/AFM199yf2/D2S2237/ 2205 9-9276-163 2q33.3 g1909/AFM199yf2/D2S2237/ 2208 9-937-125 13q31.1 g14575/D13S1196/MR8039 g8824/D13S1196/WI5275/ 2218 9-941-265 13q31.1 g14575/D13S1196/MR8039 g8824/D13S1196/WI5275/ 2222 9-942-381 13q31.1 g14575/D13S1196/MR8039 g8824/D13S1196/WI5275/ 2233 9-949-214 13q31.1 g14575/D13S1196/MR8039 g8824/D13S1196/WI5275/ 2237 9-950-418 13q31.3 g2255/AFM218yf10/D13S265 g8812/WI10332/ 2239 9-952-252 13q31.3 g2255/AFM218yf10/D13S265 g8812/WI10332/ 2243 9-954-45 13q31.3 g2255/AFM218yf10/D13S265 g8812/WI10332/ 2253 9-958-92 13q31.3 g2255/AFM218yf10/D13S265 g8812/WI10332/ 2255 9-961-150 13q31.3 g2255/AFM218yf10/D13S265 g8812/WI10332/ 2256 9-963-395 13q22.3-q31.1 g2243/AFM218xd12/D13S264 g2474/AFM240wh2/D13S170/ 2257 9-965-165 13q22.3-q31.1 g2243/AFM218xd12/D13S264 g2474/AFM240wh2/D13S170/ 2258 9-967-306 13q22.3-q31.1 g2243/AFM218xd12/D13S264 g2474/AFM240wh2/D13S170/ 2259 9-976-246 13q22.3-q31.1 g2243/AFM218xd12/D13S264 g2474/AFM240wh2/D13S170/ 2260 9-979-343 13q22.3-q31.1 g2243/AFM218xd12/D13S264 g2474/AFM240wh2/D13S170/ 2420 9-1091-446 21q22.1-q22.2 g7006/D21S211 g7724/D21S1283/ 2445 9-1105-127 13p13-q11; g9370/AFMG51E07/G51E07/ 14p13-q11.1; 15p13-q12; 21p13-q11.1; 22p13-q11.1; 3p11-q11.2; 4p11-q11; 9p11-q12 2585 9-1202-340 21q22.3 g7781/D21S1684 g7882/D21S1859 g7889/D21S1868 g7893/D21S1871 g7897/D21S1875/ 2587 9-1203-272 21q22.3 g7781/D21S1684 g7882/D21S1859 g7889/D21S1868 g7893/D21S1871 g7897/D21S1875/ 2597 9-1211-59 21q22.3 g7781/D21S1684 g7882/D21S1859 g7889/D21S1868 g7893/D21S1871 g7897/D21S1875/ 2655 9-12965-451 5q32 g1840/AFM196xc7/D5S479/ 2656 9-12969-128 5q32 g1840/AFM196xc7/D5S479/ 2657 9-12970-339 5q32 g1840/AFM196xc7/D5S479/ 2658 9-12973-162 5q32 g1840/AFM196xc7/D5S479/ 2675 9-1370-401 1q43 g401/D1S2483/G04024 g428/EST386335/R1150010/SGC35175/ 2706 9-14944-119 5q30.1-q31.2 g1446/AFM127xh4/D5S4021 2707 9-14949-472 5q31.1-q31.2 g1446/AFM127xh4/D5S4021 2708 9-15000-259 1q42.3-q43 g427/AFMa111yd5/Z67285/ 2717 9-15653-359 Xp21.3-p21.2 g3025/AFM292wb9/DXS1218/ 2718 9-15654-122 Xp21.3-p21.2 g3025/AFM292wb9/DXS1218/ 2721 9-1591-235 1q43 g1262/AFM088xe5/D1S2850 g17846/RH12368/stSG3536 g18893/RH34172/L18266/SHGC-4752 g307/WI-11654/RH50099/RH63795/R10130 g317/EST382595/RH49984/SGC34592 g402/D1S1680/G09467 g421/WI-12850/RH50542/Z41492 g4882/AFMa245wd5/D1S2678 g4901/AFMa247wg9/D1S2680/ 2725 9-16026-359 5q31.3-q32 g1166/AFM066xf11/D5S396 g20471/RH60098/WI-10312/ 2726 9-1624-377 1q43 g2259/AFM218zb6/D1S321/ 2771 9-18122-403 20p12 g1846/AFM197xb12/D20S112/ 2772 9-18126-160 20p12 g1846/AFM197xb12/D20S112/ 2773 9-18127-283 20p12 g1846/AFM197xb12/D20S112/ 2774 9-18141-152 17p12 g1854/AFM197xh6/D17S9221 2778 9-18334-485 2q35 g11465/WI-11020/EST206594/WI-11020/RH56985/ R36533 g24950/SHGC-6253/G02482 g891/EST165729/SGC33785/T95608 g892/RH56759/NIB1635/T16652 g893/WI-11020/EST206594/R36533 g894/WI-14333/EST228327/RH57030/R44333 g896/WI-22153/RH56193/ 2784 9-18645-309 2q34-q35 g2044/AFM205yb4/D2S295/ 2785 9-18696-213 17q12 g23714/RH55113/G04954/WI-5770/ 2786 9-18698-346 11q23-q24 g4760/AFMa222xc5/D11S4104/ 2787 9-18710-208 17q23-q24 g1684/AFM168xd12/D17S794/ 2788 9-18717-319 17q23-q24 g1684/AFM168xd12/D17S794/ 2789 9-18718-362 17q23-q24 g1684/AFM168xd12/D17S794/ 2793 9-18944-242 6p21.2-p21.1 g10052/AFM165YD12/ 2794 9-19023-347 6p21.2-p21.1 g10052/AFM165YD12/ 2795 9-19027-222 6p21.2-p21.1 g10052/AFM165YD12/ 2796 9-19033-208 17p13 g23660/RH54938/G05471/WI-9926/ 2839 9-19324-214 5q32 g11857/WI-12096/EST115160/WI-12096/RH59849/ T61077 g20484/RH60414/H11651/SGC32445 g2482/AFM240xg3/D5S500 g653/WI-12096/T61077/ 2840 9-19330-274 5q32 g11857/WI-12096/EST115160/WI-12096/RH59849/ T61077 g20484/RH60414/H11651/SGC32445 g2482/AFM240xg3/D5S500 245 g653/WI-12096/T61077/ 2946 9-20226-32 6p22.3 g26006/SHGC-13860/T55234/ 2947 9-20228-290 6p22.3 g26006/SHGC-13860/T55234/ 2948 9-20234-101 6p22.3 g26006/SHGC-13860/T55234/ 2953 9-20958-373 22q12.3-q13.1 g24314/RH57665/T87617/WI-20641/ 2954 9-21057-337 21q22.3 g1183/AFM071xa1/D21S1912 g7874/D21S1851 g7918/D21S1930/ 2955 9-21059-118 21q22.3 g1183/AFM071xa1/D21S1912 g7874/D21S1851 g7918/D21S1930/ 2961 9-21227-295 3p24.3-p25.1 g24997/D3S4113/ 3074 9-22202-58 11q22.3-q23.1 g22439/RH51746/WI-14282/ 3075 9-22204-391 11q22.3-q23.1 g22439/RH51746/WI-14282/ 3076 9-22206-455 11q22.3-q23.1 g22439/RH51746/WI-14282/ 3082 9-2251-151 21q22.1 g7689/D21S1230/ 3083 9-22530-48 6p22.1-p21.3 g1070/AFM031yh12/D6S258/ 3084 9-22537-280 6p22.1-p21.3 g1070/AFM031yh12/D6S258/ 3085 9-22567-243 14q24.3-q31 g23143/RH53688/G04281/WI-3377 g27731/D14S929/ 3086 9-22572-72 14q24.3-q31 g23143/RH53688/G04281/WI-3377 g27731/D14S929/ 3089 9-22729-352 2q35 g19425/RH56275/H62242/SGC32398/RH56275/ 3090 9-22768-113 2q35 g24953/SHGC-971/Z17049 g4193/AFMa109wg5/D2S2151/ 3091 9-22814-349 21q21.1 g7005/D21S172 g7814/D21S1721E/ 3092 9-22818-33 21q21.1 g7005/D21S172 g7814/D21S1721E/ 3093 9-22826-311 21q21.1 g7005/D21S172 g7814/D21S1721E/ 3095 9-23113-388 22q12;2p23 g19151/RH57021/R95095/SGC33508 g19152/RH56155/WI-10842/ 3102 9-2333-423 21q22.1 g7774/D21S1677 g7876/D21S1853 g7877/D21S1854 g7886/D21S1865 g7887/D21S1866/ 3103 9-2341-485 21q22.1 g7774/D21S1677 g7876/D21S1853 g7877/D21S1854 g7886/D21S1865 g7887/D21S1866/ 3104 9-2342-217 21q22.1 g7774/D21S1677 g7876/D21S1853 g7877/D21S1854 g7886/D21S1865 g7887/D21S1866/ 3105 9-23427-283 Xp11.22-p11.21 g4806/AFMa230vc1/DXS8032/ 3116 9-23696-164 5q31.2 g1948/AFM200ya9/D5S414 g20384/AFM240yf6 g25905/SHGC-11406/T50434 g25906/SHGC-893/Z16886/ 3117 9-23701-104 5q31.2 g1948/AFM200ya9/D5S414 g20384/AFM240yf6 g25905/SHGC-11406/T50434 g25906/SHGC-893/Z16886/ 3118 9-23702-437 5q31.2 g1948/AFM200ya9/D5S414 g20384/AFM240yf6 g25905/SHGC-11406/T50434 g25906/SHGC-893/Z16886/ 3119 9-2371-93 21q22.1 g7798/D21S1700 g7879/D21S1856/ 3120 9-23711-455 15q23 g27865/D15S1242/ 3121 9-23730-202 15q23 g27865/D15S1242/ 3188 9-24369-263 7q11.23-q21.1 g11961/WI-12513/EST25131/RH61608/R74459/ 3189 9-24397-315 11q22.3-q23.1 g15763/WI-30893/RH52168/ 3190 9-24408-202 14q24.3 g23123/RH53961/T40920/SGC32981/SHGC-32981/ G27696/G25597/EST91724 g27725/SHGC-942/Z16981/RH49039/ 3192 9-24412-279 1q24-q25 g18536/RH26850/Z38322/ 3193 9-24415-85 1q24-q25 g18536/RH26850/Z38322/ 3194 9-24470-168 Xq22.2 g24379/RH63325/G04441/WI-3796/ 3195 9-24472-179 Xq22.2 g24379/RH63325/G04441/WI-3796/ 3196 9-24480-44 Xq22.2 g24379/RH63325/G04441/WI-3796/ 3197 9-24485-55 Xp22.1 g24364/RH63434/R59327/SGC31861/ 3198 9-24490-363 Xp22.1 g24364/RH63434/R59327/SGC31861/ 3199 9-24492-351 Xp22.1 g24364/RH63434/R59327/SGC31861/ 3200 9-24581-253 22q11.2-q12 g24283/RH57762/R54799/WI-21996 g24289/RH57833/G03738/WI-373/ 3201 9-24591-33 22q11.2-q12 g24283/RH57762/R54799/WI-21996 g24289/RH57833/G03738/WI-373/ 3202 9-24592-55 4q26-q27 g20097/RH59095/H50674/WI-18054/ 3203 9-24745-413 11q23.1-q23.2 g15768 g22459/RH52046/H86791/SGC31226/ 3204 9-24753-182 11q23.1-q23.2 g15768 g22459/RH52046/H86791/SGC31226/ 3205 9-24768-233 22q13 g775/stSG5976/RH27889/ 3220 9-25362-247 17q12-q21 g2174/AFM211zd12/D17S1842/ 3223 9-25446-121 15q14 g15961/WI-7216/RH54415/G06453/G00-679-062/ UTR-03037/M99564/ 3224 9-25496-221 2q37.1 g19470/RH56828/T54332/SGC33096 g24961/SHGC-1072/Z17274/RH13453/ 3225 9-25497-242 21q22.2 g16433 g16433 g24246/RH57580/SGC32448 g28577/SHGC-10474 g28580/SHGC-10477 g7818/D21S1725E/ 3226 9-2559-253 21q22.3 g24258 g24258/RH57551/R48588/SGC34143 g24259/RH57619/H53556/SGC34732/ 3228 9-2566-112 21q22.3 g24258 g24258/RH57551/R48588/SGC34143 g24259/RH57619/H53556/SGC34732/ 3229 9-2567-329 21q22.3 g24258 G24258/RH57551/R48588/SGC34143 g24259/RH57619/H53556/SGC34732/ 3230 9-2571-242 21q22.3 g24258 g24258/RH57551/R48588/SGC34143 g24259/RH57619/H53556/SGC34732/ 3235 9-26051-273 10p12.1-p11.2 g21912/RH51110/SGC31510/WICGR/ 3236 9-26058-275 19q13.1 g3203 g3203/AFM304zg1/D19S417/ 3237 9-26074-400 6p22 g13490/WI-17546/EST261382/RH61086 g20682/RH60603/R26060/WI-11794/RH37391/ 3238 9-26076-376 6p22 g13490/WI-17546/EST261382/RH61086 g20682/RH60603/R26060/WI-11794/RH37391/ 3239 9-2630-67 21q22.2 g16433 g16433 g24246/RH57580/SGC32448 g28577/SHGC-10474 g28580/SHGC-10477 g7818/D21S1725E/ 3240 9-2633-129 21q22.2 g16433 g16433 g24246/RH57580/SGC32448 g28577/SHGC-10474 g28580/SHGC-10477 g7818/D21S1725E/ 3241 9-2634-341 21q22.2 g16433 g16433 g24246/RH57580/SGC32448 g28577/SRGC-10474 g28580/SHGC-10477 g7818/D21S1725E/ 3242 9-2636-64 21q22.2 g16433 g16433 g24246/RH57580/SGC32448 g28577/SHGC-10474 g28580/SHGC-10477 g7818/D21S1725E/ 3243 9-2642-255 21q22.2 g16433 g16433 g24246/RH57580/SGC32448 g28577/SRGC-10474 g28580/SRGC-10477 g7818/D21S1725E/ 3244 9-2645-118 21q22.1 g2908 g2908/AFM283xh9/D21S1255 g7915/D21S1928/ 3245 9-2647-368 21q22.1 g2908 g2908/AFM283xh9/D21S1255 g7915/D21S1928/ 3246 9-2649-107 21q22.1 g2908 g2908/AFM283xh9/D21S1255 g7915/D21S1928/ 3493 9-4534-158 15q14-q15 g15965/WI-11934/EST197813/RH54176/R27768 g15966/WI-19599/ 3497 9-4589-169 1q43 g314/WI-10464/ 3502 9-468-271 10p11.2 g2598 g2598/AFM254xb1/D10S224/ 3527 9-4903-395 21q21.1 g7005 g7005/D21S172 g7814/D21S1721E/ 3528 9-499-294 10p12.1-p11.2 g4171/AFMa106vf5/D10S1639/ 3532 9-5098-29 7q11.23-q21.1 g2935/AFM286xf9/D7S669/ 3534 9-5112-188 7q11.23-q21.1 g2935/AFM286xf9/D7S669/ 3545 9-5549-289 2q33 g675/RH18054/ 3546 9-5569-237 2q33 g675/RH18054/ 3547 9-5575-330 2q34-q35 g19417/RH56312/R44983/SGC31824 g646/RH56681/SGC33209/R02572 g647/D2S2635 g887/WI-10220/EST135001/T72644/ 3548 9-5602-372 2q34-q35 g19417/RH56312/R44983/SGC31824 g646/RH56681/SGC33209/R02572 g647/D2S2635 g887/WI-10220/EST135001/T72644/ 3550 9-568-101 21q22.1-q22.2 g2421/AFM238wc3/D21S267 g28563/SHGC-3796/Z17065/ 3577 9-6401-64 9q34.2 g2548/AFM248wf1/D9S179 g26813/SHGC-3659/Z17118/ 3603 9-7117-266 10q25.3-q26.1 g5771/AFMb320zb5/D10S1722/ 3604 9-7203-286 1q43 g12003/WI-12648/EST332992/RH49763/RH63832/ R92197/ 3617 9-7696-215 11q24 g5443/AFMb066zg9/D11S4144/ 3618 9-7702-225 11q24 g5443/AFMb066zg9/D11S4144/ 3619 9-7772-185 14q32.2 g27781 g27781/SHGC-1408/Z23999/ 3622 9-7860-320 2q34-q35 g2794/AFM273va9/D2S334/ 3623 9-7886-350 2q35 g2211/AFM214ye1/D2S301/ 3663 9-9316-399 2q34-q35 g12748/WI-15052/EST283445/RH56222 g19413/AFMb299wb5/ 3689 9-974-231 13q22.3-q31.1 g2243/AFM218xd12/D13S264 g2474/AFM240wh2/D13S170/ 3782 9-13794-147 1q43 g1262/AFM088xe5/D1S2850 g17846/RH12368/stSG3536 g18893/RH34172/L18266/SHGC-4752 g307/WI-11654/RH50099/RH63795/R10130 g317/EST382595/RH49984/SGC34592 g402/D1S1680/G09467 g421/WI-12850/RH50542/Z41492 g4882/AFMa245wd5/D1S2678 g4901/AFMa247wg9/D1S2680/ 3788 9-19032-132 17p13 g23660/RH54938/G05471/WI-9926/ 3794 9-21051-435 21q22.3 g1183 g1183/AFM071xa1/D21S1912 g7874/D21S1851 g7918/D21S1930/ 3817 9-22679-148 18p11.31 g23841/RH55516/G03618/WI-4219/ 3818 9-23095-184 1q21-q22 g17211/RH75/D19615/ 3819 9-23370-249 2q35 g16359/WI-19704/ 3830 9-24267-190 2q33-q34 g2141/AFM210yf10/D2S155/ 3832 9-253-97 21q22.1 g24236 g24236/RH57620/H16797/SGC32169 g2978/AFM289xh1/D21S1910/ 3889 9-5605-90 2q34-q35 g19417/RH56312/R44983/SGC31824 g646/RH56681/SGC33209/R02572 g647/D2S2635 g887/WI-10220/EST135001/T726441 3896 9-7215-279 8p21 g12037/WI-12748/RH62389/G13379/HSC25C022/ 3909 9-344-439 19q13.2-q13.3 g448/RH11470/RH1669/ 3910 9-366-274 19q13.2-q13.3 g448/RH11470/RH1669/ 3911 9-359-308 19q13.2-q13.3 g448/RH11470/RH1669/ 3912 9-355-219 19q13.2-q13.3 g448/RH11470/RH1669/ 3913 9-365-344 19q13.2-q13.3 g448/RH11470/RH1669/

TABLE 10 SEQ Marker Chromosomal ID No. Name Localization Adjacent STS 232 99-13647-278 11q12 g403/WI-7199/RH49904/M30269 g406/AFM093XG5/Z66633 g416/D1S3481/G13394 g496/WI-20654/TWIK1/EST/RH50086/T89039/ 233 99-13652-407 11q12 g403/WI-7199/RH49904/M30269 g406/AFM093XGS/Z66633 g416/D1S3481/G13394 g496/WI-20654/TWIK1/EST/RH50086/T89039/ 234 99-13663-218 11q12 g403/WI-7199/RH49904/M30269 g406/AFM093XG5/Z66633 g416/D1S3481/G13394 g496/WI-20654/TWIK1/EST/RH50086/T89039/ 235 99-13666-275 11q12 g403/WI-7199/RH49904/M30269 g406/AFM093XGS/Z66633 g416/D1S3481/G13394 g496/WI-20654/TWIK1/EST/RH50086/T89039/ 237 99-13671-396 11q12 g403/WI-7199/RH49904/M30269 g406/AFM093XG5/Z66633 g416/D1S3481/G13394 g496/WI-20654/TWIK1/EST/RH50086/T89039/ 322 99-1423-361 2p15-p14 g411/AFMa045xa9/ 326 99-1426-185 2p15-p14 g411/AFMa045xa9/ 517 99-15072-64 2q32-q33 g27160/RH11834/ 518 99-15087-77 2q32-q33 g27160/RH11834/ 550 99-1533-471 2p15-p14 g411/AFMa045xa9/ 555 99-1535-241 2p15-p14 g411/AFMa045xa9/ 556 99-1537-243 2p15-p14 g411/AFMa045xa9/ 592 99-15595-41 2q32.1-q32.2 g313/D1S3401/G04332/ 593 99-15596-64 2q32.1-q32.2 g313/D1S3401/G04332/ 594 99-15599-252 2q32.1-q32.2 g313/D1S3401/G04332/ 595 99-15605-221 2q32.1-q32.2 g313/D1S3401/G04332/ 596 99-15606-326 2q32.1-q32.2 g313/D1S3401/G04332/ 604 99-15705-110 15q23-q24 g1484/AFM143xd12/D25128/ 605 99-15717-120 15q23-q24 g1484/AFM143xd12/D2S128/ 606 99-15718-234 15q23-q24 g1484/AFM143xd12/D2S128/ 628 99-15891-215 Xq26.3 g8814/WI-14718/ 699 99-16559-90 g2862/AFM278yd1/D6S444/ 700 99-16562-182 g2862/AFM278yd1/D6S444/ 701 99-16563-263 g2862/AFM278yd1/D6S444/ 702 99-16564-118 g2862/AFM278yd1/D6S444/ 901 99-18085-94 3p23-p22 g6189/AFMc013ye9/D4S3001/ 902 99-18086-434 3p23-p22 g6189/AFMc013ye9/D4S3001/ 903 99-18087-152 3p23-p22 g6189/AFMc013ye9/D4S3001/ 923 99-18253-407 g22462/RH52139 g4351/AFMa130we1/D11S3178/ 924 99-18255-259 g22462/RH52139 g4351/AFMa130we1/D11S3178/ 929 99-18288-205 g4651/AFMa202zc1/D4S2923/ 930 99-18289-36 g4651/AFMa202zc1/D4S2923/ 932 99-18306-377 g10904/RH60078/ 933 99-18307-371 g10904/RH60078/ 934 99-18310-262 g10904/RH60078/ 935 99-18312-58 g10904/RH60078/ 964 99-18551-389 g1461/AFM136xd2/D5S403 g25942/SHGC-758/ 967 99-18582-422 g10470/D5S1491 g22231/RH52188/ 968 99-18588-175 g26653/SHGC-1005/Z17109 g3397/AFM324td5/D5S671/ 969 99-18596-83 g26653/SHGC-1005/Z17109 g3397/AFM324td5/D5S671/ 970 99-18597-415 g26653/SHGC-1005/Z17109 g3397/AFM324td5/D5S671/ 971 99-18599-347 g26653/SHGC-1005/Z17109 g3397/AFM324td5/D5S671/ 983 99-18666-483 g11811/WI-11885/ 984 99-18667-392 g11811/WI-11885/ 985 99-18669-223 g11811/WI-11885/ 996 99-18751-217 1q22 g8823/WI-4658/ 997 99-18755-267 1q22 g8823/WI-4658/ 998 99-18774-69 g11451/D5S2428/10859/ 999 99-18775-161 g11451/D5S2428/10859/ 1000 99-18777-130 g11451/D5S2428/10859/ 1001 99-18802-308 g13318/WI-16922/ 1005 99-18822-368 6p21.1-p12 g1552/AFM155ye1/D25280/ 1006 99-18826-378 6p21.1-p12 g1552/AFM155ye1/D2S280/ 1007 99-18827-92 6p21.1-p12 g1552/AFM155ye1/D2S280/ 1009 99-18847-263 6q24 g2318/AFM224zf4/D2S161/ 1010 99-18853-64 g13318/WI-16922/ 1011 99-18855-173 g13318/WI-16922/ 1012 99-18860-308 g13318/WI-16922/ 1024 99-19008-237 g3339/AFM319zf9/D2S377/ 1025 99-19013-384 g3339/AFM319zf9/D2S377/ 1026 99-19016-51 g3339/AFM319zf9/D2S377/ 1084 99-20348-403 g15529/WI-30719/RH64237/ 1086 99-20353-229 g15529/WI-30719/RH64237/ 1087 99-20357-359 g25667/SHGC-12669 g5998/AFMb349yf9/D4S2992/ 1194 99-21141-314 6q23 g1323/AFM105xc1/D2S318/105xc1/ 1195 99-21148-269 6q23 g1323/AFM105xc1/D2S318/105xc1/ 1196 99-21149-129 6q23 g1323/AFM105xc1/D25318/105xc1/ 1197 99-21167-159 6q23 g1323/AFM105xc1/D2S318/105xc1/ 1230 99-22160-331 13q22 g17798/RH12008/ 1231 99-22167-79 13q22 g17798/RH12008/ 1232 99-22172-304 13q22 g17798/RH12008/ 1234 99-22189-248 2q13-q14 g1050/AFM026wh7/D12S1595/026wh7 g28792/SHGC-37555/ 1236 99-22191-339 2q13-q14 g1050/AFM026wh7/D12S1595/026wh7 g28792/SHGC-37555/ 1237 99-22192-383 2q13-q14 g1050/AFM026wh7/D12S1595/026wh7 g28792/SHGC-37555/ 1241 99-22215-391 18p11.31 g15435/D15S1203/WI-9767 g16860/WI-20135/ 1242 99-22217-423 18p11.31 g15435/D15S1203/WI-9767 g16860/WI-20135/ 1244 99-22227-275 18p11.31 g15435/D15S1203/WI-9767 g16860/WI-20135/ 1247 99-22265-294 16 g3692/AFMa041yb5/D16S3125/ 1248 99-22266-474 16 g3692/AFMa041yb5/D16S3125/ 1251 99-22333-237 g4979/AFMa286ze9/D5S1994/ 1252 99-22336-316 g4979/AFMa286ze9/D5S1994/ 1253 99-22337-199 g4979/AFMa286ze9/D5S1994/ 1255 99-22356-370 g11856/WI-12093/ 1256 99-22357-186 g11856/WI-12093/ 1258 99-22409-141 g11856/WI-12093/ 1267 99-22490-246 13q14.1 WI-18828/ g16914 1268 99-22491-79 13q14.1 WI-18828/ g16914 1270 99-22503-146 13q14.1 WI-18828/ g16914 1271 99-22506-395 1p32.1-p31.3 g20699/RH61218/ 1272 99-22513-90 1p32.1-p31.3 g20699/RH61218/ 1273 99-22520-413 19q12-q13.1 g18667/RH29813/ 1307 99-22712-242 11q14-q21 g10031/AFM115YB6/w1773 g19151/RH57021/R95095/SGC33508/ 1308 99-22718-94 11q14-q21 g10031/AFM115YB6/w1773 g19151/RH57021/R95095/SGC33508/ 1310 99-22728-207 11q14-q21 g10031/AFM115YB6/w1773 g19151/RH57021/R95095/SGC33508/ 1322 99-22857-88 12q22 g2972/AFM289vfS/D2S346/ 1356 99-23266-146 5q13 g12748/WI-15052/EST283445/RH56222/ 1357 99-23269-263 5q13 g12748/WI-15052/EST283445/RH56222/ 1385 99-23469-288 14q31 g20668/RH60581/ 1387 99-23473-35 14q31 g20668/RH60581/ 1389 99-23488-239 2q32.3-q33 g24198/RH57320/SGC30627/ 1390 99-23492-151 2q32.3-q33 g24198/RH57320/SGC30627/ 1391 99-23496-94 2q32.3-q33 g24198/RH57320/SGC30627/ 1392 99-23510-45 8q21.1-q21.2 g6072/AFMb359wh1/D11S4179/ 1393 99-23528-452 8q21.1-q21.2 g6072/AFMb359wh1/D11S4179/ 1426 99-24381-217 4q34-q35 g13574/WI-17820/ 1427 99-24385-210 4q34-q35 g13574/WI-17820/ 1428 99-24388-391 4q34-q35 g13574/WI-17820/ 1437 99-24438-402 10p12-p11.2 g15056/WI-7090 g18656/RH29333 g3307/AFM317yc5/D7S685/ 1438 99-24441-431 10p12-p11.2 g15056/WI-7090 g18656/RH29333 g3307/AFM317ycS/D7S685/ 1459 99-25005-154 6p25 g1147/AFM059yg5/D4S2988/059yg5/ 1460 99-25007-131 6p25 g1147/AFM059yg5/D4S2988/059yg5/ 1465 99-25129-166 g6011/AFMb351xf9/D5S2050/ 1466 99-25134-296 g6011/AFMb351xf9/D5S2050/ 1473 99-25379-389 g2028/AFM205wh8/D5S417/ 1474 99-25382-226 g2028/AFM205wh8/D5S417/ 1475 99-25387-220 g2028/AFM205wh8/D5S417/ 1476 99-25400-379 g25712/SHGC-1789 g25713/SHGC-6395 g2753/AFM268zd9/D5S630/ 1477 99-25412-354 g11977/WI-12573 g13670/WI-18094/ 1485 99-25458-103 14q31 g13865/WI-18706/ 1486 99-25503-333 5q22-q23.1 g6127/AFMc003zg5/D3S3681/ 1487 99-25507-373 15q15 g19212/RH57152 g20451/RH59692/ 1488 99-25510-390 15q15 g19212/RH57152 g20451/RH59692/ 1492 99-25575-303 g15433/D3S3958/WI-9747 g6236/AFMc024yd1/D3S3692/ 1493 99-25618-196 g10319/AFMB346XE9/w2056 g4935/AFMa275zh1/D3S3588/ 1494 99-25620-360 g10319/AFMB346XE9/w2056 g4935/AFMa275zh1/D3S3588/ 1495 99-25629-262 g10319/AFMB346XE9/w2056 g4935/AFMa275zh1/D3S3588/ 1496 99-25657-314 g5177/AFMa348yd9/D4S2955/ 1497 99-25672-97 g10965/RH59124/ 1498 99-25676-211 g10965/RH59124/ 1499 99-25678-307 g10965/RH59124/ 1501 99-25712-418 g11689/WI-11614 g13669/WI-18093/ 1504 99-25725-80 g14391/D4S2653/WI-4583/ 1505 99-25732-152 g14391/D4S2653/WI-4583/ 1506 99-25745-36 g11645/WI-11521 g22536/RH52006/WI-6398/ 1573 99-477-302 11q22.3-q23.1 g5966/AFMb345ya9/D10S1732/ 1577 99-482-130 11q22.3-q23.1 g5966/AFMb345ya9/D10S1732/ 1580 99-483-424 11q22.3-q23.1 g5966/AFMb345ya9/D10S1732/ 1585 99-486-243 11q22.3-q23.1 g5966/AFMb345ya9/D10S1732/ 1592 99-4924-254 3q13.3 g1572/AFM157xg9/D2S2178/ 1593 99-4928-102 3q13.3 g1572/AFM157xg9/D2S2178/ 1778 99-6327-270 7q31.1 g5753/AFMb319ze5/D4S2974/ 1779 99-6332-143 7q31.1 g5753/AFMb319ze5/D4S2974/ 1786 99-6415-279 9q33 g407/AFM151XB8/Z66679/ 1787 99-6421-210 9q33 g407/AFM151XB8/Z66679/ 1788 99-6423-90 9q33 g407/AFM151XB8/Z66679/ 1789 99-6426-413 9q33 g407/AFM151XB8/Z66679/ 1790 99-6427-190 9q33 g407/AFM151XB8/Z66679/ 1800 99-6478-358 8q13 g775/stSG5976/RH27889/ 1801 99-6480-440 8q13 g775/stSG5976/RH27889/ 1802 99-6489-237 8q13 g775/stSG5976/RH27889/ 1887 99-7129-335 11q21 g1453/AFM135xf12/D2S2396/ 1888 99-7131-259 11q21 g1453/AFM135xf12/D2S2396/ 1889 99-7136-329 11q21 g1453/AFM135xf12/D2S2396/ 1890 99-7137-420 11q21 g1453/AFM135xf12/D2S2396/ 1891 99-7140-355 11q21 g1453/AFM135xf12/D2S2396/ 2104 99-8546-116 12q22 g2972/AFM289vf5/D2S346/ 2105 99-8571-396 12q15 g1392/AFM119xc7/D2S126/ 2106 99-8575-401 12q15 g1392/AFM119xc7/D2S126/ 2107 99-8576-321 12q15 g1392/AFM119xc7/D2S126/ 2108 99-8578-407 12q15 g1392/AFM119xc7/D2S126/ 2109 99-8581-443 12q15 g1392/AFM119xc7/D2S126/ 2190 99-913-140 7p22 g2474/AFM240wh2/D13S170/ 2709 99-15067-278 2q32-q33 g27160/RH11834/ 2716 99-15615-368 2q32.1-q32.2 g313/D1S3401/G04332/ 2729 99-16284-389 Xq26.3 g8814/WI-14718/ 2776 99-18321-371 g2237/AFM217yel/D5S627 g4093/AFMa084zc1/D5S2113/ 2782 99-18576-182 g10470/D5S1491 g22231/RH52188/ 2783 99-18581-34 g10470/D5S1491 g22231/RH52188/ 2790 99-18771-300 g11451/D5S2428/10859/ 2956 99-21110-304 g3373/AFM323vc1/D11S1348/ 2957 99-21123-62 g3373/AFM323vc1/D11S1348/ 2958 99-21133-169 g3373/AFM323vc1/D11S1348/ 3071 99-22181-171 4q23-q24 g20693/RH61180/ 3072 99-22187-261 13q13 g15285/D1S3356/WI-8997 g19004/RH35464/ 3073 99-22190-369 2q13-q14 g1050/AFM026wh7/D12S1595/026wh7 g28792/SHGC-37555/ 3077 99-22213-333 18p11.31 g15435/D15S1203/WI-9767 g16860/WI-20135/ 3078 99-22355-213 g11856/WI-12093/ 3087 99-22593-64 g11914/WI-12390 g12081/WI-12941 g13404/WI-17166/ 3088 99-22706-367 11q14-q21 g10031/AFM115YB6/w1773 g19151/RH57021/R95095/SGC33508/ 3094 99-22851-121 12q22 g2972/AFM289vf5/D25346/ 3096 99-23188-227 6p22.1-p21.3 g19258/RH56674/ 3097 99-23240-326 1q43;Xq25 g18121/RH16765 g24533/RH63274/WI-9960 g28803/SHGC-16321 g495/WI-20605/RH50893/ 3098 99-23246-66 6q27 g1752/AFM184xb10/D8S1178/ 3099 99-23248-308 6q27 g1752/AFM184xb10/D8S1178/ 3100 99-23249-262 6q27 g1752/AFM184xb10/D8S1178/ 3101 99-23274-182 2q33-q34 g11967/WI-12530/ 3219 99-25020-395 7q31.3 g10580/D6S1053/ 3221 99-25394-261 g25712/SHGC-1789 g25713/SHGC-6395 g2753/AFM268zd9/D5S630/ 3222 99-25406-54 g11977/WI-12573 g13670/WI-18094/ 3227 99-25654-281 g5177/AFMa348yd9/D4S2955/ 3231 99-25738-218 g11645/WI-11521 g22536/RH52006/WI-6398/ 3517 99-480-373 11q22.3-q23.1 g5966/AFMb345ya9/D10S1732/ 3575 99-6173-229 11q13.3;3q22;4 g8817/WI-3139/ p16;7p22;8p23. 1 3620 99-7815-70 9q34.1 g316/EST47321/D19656/RH50110/SGC32758/ 3621 99-7818-342 9q34.1 g316/E5T47321/D19656/RH50110/SGC32758/ 3816 99-22594-395 g11914/WI-12390 g12081/WI-12941 g13404/WI-17166/

TABLE 11 SEQ ID No Marker Name Localization 1 99-109-224 8p23 4 99-1151-516 6p21.3-p21.2 10 99-1233-183 8p23 14 99-12503-44 8p23 15 99-12504-402 8p23 16 99-12505-374 8p23 17 99-12506-199 8p23 18 99-12509-423 8p23 19 99-12513-146 8p23 20 99-12514-170 8p23 21 99-12515-205 8p23 22 99-12516-524 8p23 23 99-12518-325 8p23 24 99-12523-255 8p23 25 99-12525-277 8p23 26 99-12526-317 8p23 27 99-12527-292 8p23 28 99-12531-30 8p23 29 99-12532-199 8p23 30 99-12534-207 8p23 31 99-12535-362 8p23 32 99-12537-340 8p23 33 99-12538-142 8p23 34 99-12539-287 8p23 35 99-12540-426 8p23 36 99-12541-307 8p23 37 99-12545-121 8p23 38 99-12548-88 8p23 39 99-12558-167 8p23 40 99-12562-291 8p23 41 99-12564-354 8p23 42 99-12565-273 8p23 43 99-12575-248 8p23 44 99-12576-325 8p23 45 99-12580-268 8p23 46 99-12585-85 8p23 47 99-12593-103 8p23 48 99-12600-283 8p23 49 99-12608-71 8p23 50 99-12610-106 8p23 51 99-12611-311 8p23 52 99-12613-366 8p23 53 99-12615-235 8p23 54 99-12617-412 8p23 55 99-12618-211 8p23 56 99-12619-367 8p23 57 99-12621-114 8p23 58 99-12624-61 8p23 60 99-12632-165 8p23 61 99-12637-62 8p23 62 99-12639-311 8p23 63 99-12640-179 8p23 64 99-12650-200 8p23 65 99-12651-297 8p23 66 99-12652-459 8p23 67 99-12654-278 8p23 68 99-12656-303 8p23 69 99-12658-206 8p23 70 99-12661-92 8p23 71 99-12668-329 8p23 72 99-1268-177 1q43 73 99-12733-366 8p23 74 99-12738-57 8p23 75 99-12740-354 8p23 76 99-12749-286 8p23 77 99-12750-369 8p23 78 99-12751-406 8p23 79 99-12755-421 8p23 80 99-12756-344 8p23 81 99-12757-240 8p23 82 99-12759-420 8p23 83 99-12777-71 8p23 84 99-12782-76 8p23 85 99-12794-299 8p23 86 99-128-60 8p23 87 99-12816-101 8p23 88 99-12817-358 8p23 89 99-12819-165 8p23 90 99-12826-408 8p23 91 99-12831-345 8p23 92 99-12836-387 8p23 93 99-12842-305 8p23 94 99-12843-337 8p23 95 99-12844-130 8p23 96 99-12847-37 8p23 97 99-12848-204 8p23 98 99-12852-260 8p23 99 99-12856-183 8p23 100 99-12878-291 8p23 101 99-12880-282 8p23 102 99-12884-248 8p23 103 99-12885-261 8p23 104 99-12898-364 8p23 105 99-12899-307 8p23 106 99-1290-291 1q43 107 99-12900-165 8p23 108 99-12901-316 8p23 109 99-12903-381 8p23 110 99-12907-295 8p23 111 99-12908-369 8p23 112 99-12913-197 8p23 113 99-12914-227 8p23 114 99-12924-273 8p23 115 99-12925-487 8p23 116 99-12926-332 8p23 117 99-12931-173 8p23 118 99-12948-61 8p23 119 99-12952-199 8p23 120 99-12956-43 8p23 121 99-12957-448 8p23 122 99-12961-318 8p23 123 99-12962-181 8p23 124 99-12963-255 8p23 125 99-12964-230 8p23 189 99-1342-51 1q43 195 99-1346-503 1q43 202 99-1351-264 1q43 214 99-1356-500 1q43 219 99-1359-355 8p23.2-23.1 228 99-1362-126 8p23.2-23.1 291 99-1404-135 1q43 341 99-14385-117 3q27 342 99-14392-431 3q27 343 99-14393-190 3q27 345 99-14405-105 3q27 358 99-14553-224 1q42.3 359 99-14562-402 1q42.3 360 99-14566-320 1q42.3 361 99-14574-310 1q42.3 362 99-14581-365 8p23 363 99-14591-172 8p23 364 99-14595-210 8p23 365 99-14596-174 8p23 366 99-14597-85 8p23 367 99-14598-91 8p23 368 99-14599-220 8p23 369 99-14600-207 8p23 370 99-14601-448 8p23 371 99-14607-267 8p23 372 99-14609-467 8p23 373 99-14610-351 8p23 374 99-14611-241 8p23 375 99-14612-100 8p23 376 99-14614-248 8p23 377 99-14615-65 8p23 378 99-14616-35 8p23 379 99-14618-147 8p23 380 99-14619-325 8p23 382 99-14620-253 8p23 383 99-14621-96 8p23 384 99-14622-276 8p23 385 99-14626-307 8p23 386 99-14627-272 8p23 387 99-14628-312 8p23 388 99-14629-274 8p23 389 99-14630-75 8p23 390 99-14634-350 8p23 391 99-14635-296 8p23 392 99-14637-366 8p23 393 99-14638-276 8p23 394 99-14643-27 8p23 395 99-14644-395 8p23 396 99-14647-227 8p23 397 99-14651-205 8p23 398 99-14652-120 8p23 399 99-14653-138 8p23 400 99-14622-352 8p23 401 99-14664-289 8p23 402 99-14665-199 8p23 403 99-14669-238 8p23 404 99-14671-175 8p23 405 99-14676-313 8p23 406 99-14677-358 8p23 407 99-14678-75 8p23 408 99-14679-241 8p23 411 99-14690-84 8p23 412 99-14692-46 8p23 413 99-14699-149 8p23 414 99-147-181 8p23.3-p23.2 415 99-14701-264 8p23 416 99-14704-59 8p23 417 99-14708-142 8p23 419 99-14710-107 8p23 420 99-14712-163 8p23 421 99-14714-237 8p23 422 99-14717-132 8p23 424 99-14722-272 8p23 425 99-14729-284 8p23 426 99-14733-26 8p23 427 99-14735-328 8p23 429 99-14746-377 8p23 430 99-14753-194 8p23 431 99-14756-270 8p23 433 99-14761-194 8p23 434 99-14773-383 8p23 435 99-14776-79 8p23 436 99-14777-100 8p23 437 99-14782-152 8p23 438 99-14784-212 8p23 439 99-14785-92 8p23 440 99-14786-59 8p23 441 99-1479-158 8p23 442 99-14792-43 8p23 443 99-14796-227 8p23 444 99-14799-57 8p23 445 99-148-182 8p23.3-p23.2 446 99-1480-290 8p23 447 99-14802-60 8p23 448 99-14803-157 8p23 449 99-14804-216 8p23 450 99-14805-58 8p23 451 99-14806-108 8p23 452 99-14807-150 8p23 453 99-1481-285 8p23 454 99-14810-407 8p23 455 99-14812-189 8p23 456 99-14817-323 8p23 457 99-14818-430 8p23 458 99-14819-278 8p23 459 99-14820-76 8p23 460 99-14821-48 8p23 461 99-14826-238 8p23 462 99-14828-214 8p23 463 99-14833-226 8p23 464 99-1484-328 8p23 465 99-14843-195 8p23 466 99-14844-143 8p23 467 99-1485-251 8p23 468 99-14850-136 8p23 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99-23995-407 4q25 3149 99-24000-316 4q25 3150 99-24003-172 4p16 3151 99-24004-200 4p16 3152 99-24007-362 4p16 3153 99-24020-379 4 3154 99-24038-103 4 3155 99-24063-363 4 3156 99-24073-384 4 3157 99-24075-45 4 3158 99-24079-268 4 3159 99-24084-110 4 3160 99-24092-209 4 3161 99-24096-386 4 3162 99-24105-247 4 3163 99-24113-332 4 3164 99-24117-169 4 3165 99-24119-368 4 3166 99-24123-125 4 3167 99-24140-394 4 3168 99-24148-332 4 3169 99-24152-268 4 3170 99-24155-271 4 3171 99-24156-107 4 3172 99-24167-85 4 3173 99-24175-218 4 3174 99-24180-390 4 3175 99-24182-326 4q25 3176 99-24185-446 4q25 3177 99-24187-142 4q25 3178 99-24190-231 4q25 3179 99-24202-433 4q25 3180 99-24204-486 4 3181 99-24208-292 4 3182 99-24210-111 4 3183 99-24217-206 4 3184 99-24225-439 4 3185 99-24228-386 4 3186 99-24232-419 4 3187 99-24234-352 4 3191 99-2441-512 1q21.1-q21.2 3206 99-24855-180 6q21 3207 99-24863-199 6q21 3208 99-24867-219 6q27 3209 99-24871-435 6q27 3210 99-24889-311 4 3211 99-24897-276 4q25 3212 99-24904-187 4q25 3213 99-24909-440 22q11.2 3214 99-24917-250 7q22-q31 3215 99-24930-299 17 3216 99-24936-332 17 3217 99-24965-416 11p15.5 3218 99-24966-423 11p15.5 3251 99-2726-364 21 3252 99-2734-400 21q22.2 3253 99-2740-351 17 3254 99-2752-213 17 3255 99-2760-182 21 3256 99-2761-223 21 3257 99-2765-279 21 3258 99-2790-217 21 3259 99-2797-399 21 3260 99-2816-62 21 3261 99-2817-88 21 3262 99-2819-108 21 3263 99-2820-199 21 3264 99-2832-152 21 3267 99-2881-61 22q12 3268 99-2895-47 7q21-7q22 3269 99-2903-265 7q21-7q22 3270 99-2906-80 7q21-7q22 3271 99-2914-48 7q21-7q22 3272 99-2922-171 7q21-7q22 3273 99-2924-183 7q21-7q22 3274 99-2926-184 7q21-7q22 3275 99-2928-52 7q21-7q22 3276 99-2938-83 7q31.2 3277 99-2943-230 13q12-13q13 3278 99-2944-351 13q12-13q13 3279 99-295-355 8 3280 99-2954-160 3p21.3 3281 99-2956-239 3p21.3 3282 99-2970-318 7q21-7q22 3283 99-2978-135 7q21-7q22 3284 99-2981-53 7q21-7q22 3285 99-2988-243 7q31-q32 3286 99-2989-345 7q31-q32 3287 99-2991-256 7q31-q32 3288 99-2995-168 7q31-q32 3289 99-2999-371 7q31-q32 3290 99-3013-250 Xq23 3291 99-3018-50 Xq23 3292 99-3019-316 Xq23 3293 99-3020-369 Xq23 3294 99-3021-290 Xq23 3295 99-3044-216 5p15.2 3296 99-3045-108 5p15.2 3297 99-3046-91 5p15.2 3298 99-3047-395 5p15.2 3299 99-3058-420 7q22 3301 99-3061-369 7q22 3302 99-3106-272 7q22 3303 99-3108-156 7q22 3304 99-3109-402 7q22 3305 99-3110-321 7q22 3306 99-312-311 8q24 3307 99-3129-113 19 3308 99-3132-158 19 3309 99-3144-112 19 3310 99-3147-24 19 3311 99-3153-190 19 3312 99-3154-110 19 3313 99-3156-251 19 3314 99-3167-227 19 3315 99-3195-71 10q25-qter 3316 99-3217-274 Xp22.1 3317 99-3224-232 Xp22.1 3318 99-3231-109 Xp22.1 3319 99-3234-274 Xp22.1 3320 99-325-226 8q24.3 3321 99-3266-193 4p16.3 3322 99-3276-195 22q11.2-qter 3323 99-3279-337 4p16.3 3324 99-3293-300 4p16.3 3325 99-3296-101 4p16.3 3326 99-3299-211 4p16.3 3327 99-3305-272 22q11.2-qter 3328 99-3335-53 4p16.3 3329 99-3337-294 4p16.3 3330 99-3342-103 4p16.3 3331 99-3347-226 22q11.2-qter 3332 99-3349-124 22 3333 99-3353-350 22q11.2-qter 3334 99-3356-345 22q11.2-qter 3335 99-3368-277 4p16.3 3336 99-3373-253 4p16.3 3337 99-3374-274 4p16.3 3338 99-3385-197 16p13.3 3339 99-3390-328 16p13.3 3340 99-3391-160 16p13.3 3341 99-3393-245 22q11.2-qter 3342 99-3398-196 4p16.3 3343 99-3399-449 4p16.3 3344 99-3400-83 4p16.3 3345 99-3414-112 22q11.2-qter 3346 99-3415-215 22q11.2-qter 3347 99-3426-270 4p16.3 3348 99-3428-366 4p16.3 3349 99-3445-239 22q11.2-qter 3350 99-3453-138 22q11.2-qter 3351 99-3460-337 22q12 3352 99-3462-117 13q12-13 3353 99-3468-272 13q12-13 3354 99-3469-313 13q12-13 3355 99-3473-309 13q12-13 3356 99-3474-272 13q12-13 3357 99-3478-199 22 3358 99-3479-293 22 3359 99-3482-225 22q11.2-qter 3360 99-3483-252 22q11.2-qter 3361 99-3485-245 22q11.2-qter 3362 99-3511-130 4p16.3 3363 99-3519-374 13q12-13 3364 99-3522-210 13q12-13 3365 99-3523-270 13q12-13 3366 99-3524-403 13q12-13 3367 99-3542-336 13q12-13 3368 99-3556-129 13q12-13 3369 99-3563-121 22 3370 99-3580-122 22 3371 99-3588-188 22q12 3372 99-3589-203 22q12-qter 3373 99-3596-147 22q12 3374 99-36-69 1q23 3375 99-3601-226 22q12 3376 99-3603-80 22q12 3377 99-3604-91 22q12 3378 99-3619-330 22q12 3379 99-3620-314 22q12 3380 99-3628-31 6p21.3 3381 99-3629-219 6p21.3 3382 99-3631-159 6p21.3 3383 99-3638-259 22 3384 99-3641-230 22 3385 99-3666-280 16p13.3 3386 99-3667-190 16p13.3 3387 99-3677-196 22q11.2-qter 3388 99-3680-274 22q11.2-qter 3389 99-3689-50 22q12 3390 99-3690-355 22q12 3391 99-3699-230 22q12 3392 99-3702-226 22q12 3393 99-3703-331 22q12 3394 99-3705-195 X 3395 99-3709-366 X 3396 99-3717-68 22 3397 99-3728-341 11 3398 99-3739-215 22 3399 99-3746-337 6p21.3 3400 99-3749-174 6p21.3 3401 99-3752-210 6p21.3 3402 99-3760-59 6q21 3403 99-3761-329 6q21 3404 99-3764-198 6q21 3405 99-3765-279 6q21 3407 99-3773-337 6p21.3 3408 99-3774-351 6p21.3 3409 99-3775-98 6p21.3 3410 99-3778-97 13q12-13 3411 99-3789-293 3q12-13 3412 99-3792-294 16p13.3 3413 99-3802-197 13q12-13 3414 99-3805-125 13q12-13 3415 99-3812-243 22 3416 99-3813-122 22 3417 99-3857-261 17q22-q24 3418 99-3862-153 17q22-q24 3419 99-3875-138 8p12-q11.2 3420 99-3888-309 Xp21.2 3421 99-3893-108 12q13 3424 99-3953-77 19q13.2-13.3 3425 99-3954-362 19q13.2-13.3 3426 99-3978-146 X 3427 99-3981-156 X 3428 99-3992-185 17q21 3429 99-4001-330 20q13.2-qter 3430 99-4009-232 4q11-22 3431 99-4025-300 12pter-p12 3432 99-4052-415 6p25-p24 3433 99-4064-346 Xp21.3-p21.1 3434 99-4065-20 11p15.5 3435 99-4073-307 11p15.5 3436 99-4076-255 22q11 3437 99-4077-230 22q11 3438 99-4078-212 22q11 3439 99-4079-389 22q11 3440 99-4119-307 10 3441 99-4120-253 10 3442 99-4122-23 16p13.13-13.2 3443 99-4125-192 16p13.13-13.2 3445 99-4138-360 12q24-qter 3446 99-4139-128 12q24-qter 3447 99-4140-254 12q24-qter 3448 99-4182-113 12p13 3449 99-4193-384 1 3450 99-4194-336 1 3451 99-4199-339 4q 3452 99-4201-501 4q 3453 99-4202-223 11p15 3454 99-4203-110 11p15 3455 99-4207-210 22q12 3456 99-4218-24 22q12 3457 99-4220-241 22q12 3458 99-4225-339 X 3459 99-4231-139 17p13.3 3460 99-4232-105 17p13.3 3461 99-4233-261 12p12-13 3462 99-4238-181 Xp21.2 3463 99-4251-311 12p12-13 3466 99-4283-257 19q13.1 3467 99-4284-200 12p12-p13 3468 99-4285-370 12p12-p13 3469 99-4290-131 12p12-13 3470 99-4293-344 12p12-13 3471 99-4296-156 8q24 3472 99-4312-338 4q11-12 3473 99-4323-311 5q33.3-q34 3474 99-4325-87 11q24 3475 99-4332-136 3p14.2 3476 99-4335-371 3p14.2 3477 99-4336-171 3p14.2 3478 99-4337-369 3p14.2 3479 99-4339-180 3p14.2 3481 99-4364-360 Xp22 3482 99-4398-167 13q12-13q13 3483 99-4399-228 13q12-13q13 3484 99-4404-384 Xp22.1-22.2 3485 99-4406-115 Xp22.1-22.2 3486 99-4435-203 12p13 3487 99-4448-174 16p13.11 3488 99-4455-357 16p13.11 3489 99-4458-59 16p13.11 3490 99-4467-39 16p13 3491 99-4468-130 16p13 3492 99-4483-333 12p15 3494 99-4567-424 19 3495 99-4575-226 19 3496 99-4580-296 19 3535 99-515-151 21q22 3540 99-5329-269 6p12 3541 99-5339-196 6p12 3542 99-5347-394 1q43 3543 99-5397-353 1q43 3602 99-7086-91 10q26.2 3638 99-824-359 13 3647 99-882-250 1q23 3648 99-887-344 1q23 3650 99-892-77 1q23 3653 99-896-83 1q23 3654 99-899-252 6 3662 99-9308-416 5q31.3 3669 99-9607-402 1 3670 99-9620-241 1 3671 99-9623-330 1 3672 99-9633-32 1 3673 99-9636-423 1 3674 99-9658-42 1p35.2-36.13 3675 99-9662-213 1p35.2-36.13 3676 99-9666-363 1p35.2-36.13 3677 99-9668-185 1p35.2-36.13 3678 99-9680-363 1q23-25 3679 99-9696-292 1q24 3680 99-9697-375 1q24 3681 99-9700-289 1q24 3682 99-9704-445 1q24 3683 99-9706-448 1q24 3684 99-9709-115 1q24 3685 99-9710-242 1q24 3686 99-9714-302 1p36.13-36.22 3687 99-9717-449 1p36.13-36.22 3688 99-9726-190 1p36.21 3690 99-9745-284 1q23-24 3691 99-9751-134 1q24-q25 3692 99-9765-237 1p36.12-36.13 3693 99-9774-392 1p36.12-36.13 3694 99-9778-360 1p36.12-36.13 3695 99-9781-174 1p36.12-36.13 3696 99-9785-141 1q23.1-24.3 3697 99-9810-257 1p36.2-36.3 3698 99-9811-369 1p36.2-36.3 3699 99-9820-483 1p36.2-36.3 3700 99-9822-257 1p36.2-36.3 3701 99-9829-367 1q24-1q25 3702 99-983-278 11 3703 99-9832-128 1q24-1q25 3704 99-9833-167 1q24-1q25 3705 99-9835-217 1q24-1q25 3706 99-9837-275 1q23-1q24 3707 99-9839-416 1q23-1q24 3708 99-9840-192 1q23-1q24 3709 99-9847-25 2q1 3710 99-9849-291 2q1 3711 99-9852-276 2q1 3712 99-9854-316 2q1 3713 99-9856-252 3 3714 99-9859-132 3 3715 99-9866-365 3 3716 99-990-356 11 3717 99-9906-280 4q25 3718 99-9908-423 4q25 3719 99-991-157 16p13.11 3720 99-9915-281 4q25 3721 99-9920-245 4q25 3722 99-9921-365 4q25 3723 99-9922-154 4q25 3724 99-9926-454 4q25 3725 99-9928-454 4q25 3726 99-9929-144 4q25 3727 99-9935-418 4q25 3728 99-9941-426 4q25 3729 99-995-251 16p13.11 3730 99-996-210 16p13 3731 99-9986-202 4q25 3732 99-9988-111 4q25 3733 99-9994-226 4q25 3734 99-9995-50 4q25 3735 99-10069-366 4q25 3736 99-10074-266 4q25 3737 99-10129-177 4q25 3738 99-10198-271 4q25 3739 99-10306-345 5q 3740 99-10307-115 5q 3741 99-10326-149 6p22.2-22.3 3742 99-10393-179 6p22.3-24.1 3743 99-10685-454 6p24 3744 99-10857-217 7q31 3745 99-10948-281 7p15-p21 3746 99-11104-329 7p15-p21 3747 99-11116-199 8q21 3748 99-11117-282 8q21 3749 99-11121-461 8q21 3750 99-11124-363 8q21 3751 99-11172-373 11p11.2 3752 99-11206-379 11p14.3 3753 99-11303-223 15 3754 99-11307-168 15 3755 99-11325-188 16p11.2 3756 99-11365-273 16p11.2-p12 3757 99-11389-268 6p12.2-p12 3758 99-11395-376 16p12.2-p12 3759 99-11500-50 16p13.2-13.3 3760 99-11571-88 17 3761 99-11710-452 17 3762 99-1173-208 17 3763 99-11735-215 17 3764 99-11864-218 22 3765 99-1187-293 6 3766 99-11872-228 22 3767 99-11878-212 22q12-13 3768 99-11905-202 22q12-qter 3769 99-11932-48 22q12.1 3770 99-11964-158 22 3771 99-12164-412 X 3772 99-12227-278 Xq23 3773 99-12417-447 Xq22 3774 99-12459-119 Xq28 3775 99-12521-212 8p23 3776 99-12569-95 8p23 3777 99-1298-430 X 3778 99-1315-105 X 3783 99-14899-215 8p23.1 3789 99-19212-369 1q32.1-32.3 3790 99-19273-219 1p36.1-36.2 3791 99-19279-356 1q32.1-32.3 3792 99-19541-172 7p21 3793 99-19552-214 7p21-p22 3795 99-21246-20 1q24 3796 99-21387-465 22q11.2 3797 99-21407-352 22q11.2 3798 99-21418-83 22q11.2 3799 99-21419-85 22q11.2 3800 99-21430-308 22q11.2 3801 99-21435-96 22q11.2 3802 99-21446-240 22q11.2 3803 99-21452-173 22q11.2 3804 99-21488-376 17 3805 99-21489-227 17 3806 99-21496-248 17 3807 99-21519-446 17 3808 99-21618-178 22q11.2 3809 99-21725-371 21q22.3 3810 99-21773-155 22q12 3811 99-21781-252 22q12 3812 99-21820-230 22q13.31-13.32 3813 99-21822-50 22q13.31-13.32 3814 99-21939-170 20q12-13.12 3815 99-22404-59 16p13.11 3820 99-23568-395 10 3821 99-23824-339 12q13.1 3822 99-23969-316 12 3823 99-24032-138 4 3824 99-24048-286 4 3825 99-24074-190 4 3826 99-24082-408 4 3827 99-24104-308 4 3828 99-24138-224 4 3829 99-24172-116 4 3831 99-24949-289 17 3833 99-2694-411 1q43 3834 99-2697-336 1q43 3836 99-2851-105 1p13.3 3837 99-2889-197 22q13 3838 99-3072-323 7q21-7q22 3839 99-3089-49 7q21-7q22 3840 99-3157-203 19 3841 99-3210-341 Xp22.1 3842 99-3218-344 Xp22.1 3843 99-3251-254 4p16.3 3844 99-3298-158 4p16.3 3845 99-3300-433 22q11.2-qter 3846 99-3364-247 22q11.2-qter 3847 99-3427-271 4p16.3 3848 99-3484-96 22q11.2-qter 3849 99-3537-196 22q12 3850 99-3568-156 22q12 3851 99-3592-325 22q12-qter 3852 99-3602-245 22q12 3853 99-3608-264 22q11.2-qter 3854 99-3643-352 9q34 3855 99-3770-363 6p21.3 3856 99-3772-266 6p21.3 3857 99-3790-361 13q12-13 3858 99-3818-255 22 3859 99-3863-328 7q22-q24 3860 99-3879-245 17q11.2 3861 99-3882-312 17q11.2 3862 99-3883-329 17q11.2 3863 99-3884-355 17q11.2 3864 99-3894-333 12q13 3865 99-3936-352 Xq27.3-q28 3867 99-4029-174 12pter-p12 3869 99-4102-109 9q34 3870 99-4110-180 21q22.1 3871 99-4111-259 21q22.1 3872 99-4126-366 16p13.13-13.2 3873 99-4157-72 16p11.1 3874 99-4228-168 17p13.3 3875 99-4239-328 Xp21.2 3876 99-4254-307 7q11.23 3878 99-4311-146 4q11-12 3879 99-4381-385 Xq22.1-22.2 3880 99-4403-194 Xp22.1-22.2 3881 99-4524-296 8 3882 99-4582-359 19 3899 99-889-153 1q23 3901 99-9609-220 1p36.2-36.3 3902 99-9612-324 1p36.2-36.3 3903 99-9616-136 1p36.2-36.3 3904 99-9683-49 1q23-25 3905 99-9907-88 4q25 3906 99-993-218 16p13.11 3907 99-24069-351 4 3908 99-3855-279 17q22-q24 3915 99-123-381 8p23 3916 4-26-29 8p23 3917 4-14-240 8p23 3918 4-77-151 8p23 3919 99-217-277 8p23 3920 4-67-40 8p23 3921 99-213-164 8p23 3922 99-221-377 8p23 3924 99-1482-32 8p23 3925 4-73-134 8p23 3926 4-65-324 8p23 3927 10-32-357 13q13 3928 10-33-175 13q13 3929 10-33-234 13q13 3930 10-33-327 13q13 3931 10-35-358 13q13 3932 10-35-390 13q13 3933 10-36-164 13q13 3934 10-204-326 13q13

SEQUENCE LISTING The patent contains a lengthy “Sequence Listing” section. A copy of the “Sequence Listing” is available in electronic form from the USPTO web site (http://seqdata.uspto.gov/sequence.html?DocID=06537751B1). An electronic copy of the “Sequence Listing” will also be available from the USPTO upon request and payment of the fee set forth in 37 CFR 1.19(b)(3). 

What is claimed is:
 1. A method of determining whether an individual is at an increased risk for developing Alzheimer's disease comprising: (a) obtaining a biological sample comprising a nucleic acid from said individual; (b) determining the identity of a biallelic marker at position 24 of SEQ ID NO: 3913 within said biological sample; (c) evaluating whether an individual is at increased risk of Alzheimer's disease; wherein the presence of a T at position 24 of SEQ ID NO: 3913 is indicative of an increased risk to Alzheimer's disease.
 2. A method according to claim 1, wherein the identity of the nucleotides at said biallelic marker is determined for both copies of said biallelic marker present in said subject's genome.
 3. A method according to claim 1, further comprising amplifying a portion of said sequence comprising the biallelic marker prior to said determining step.
 4. A method according to claim 3, wherein said amplifying is performed by PCR.
 5. A method according to claim 3, wherein said determining is performed by a hybridization assay, a sequencing assay, a microsequencing assay, or an enzyme-based mismatch detection assay. 